Qualitative and quantitative analysis and their methods. What is the difference between qualitative and quantitative PCR? How is an infectious disease test done?

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We are pleased to welcome you to educational service and hope so that we can answer all your questions. Have you looked at our website in order to find out what is qualitative analysis and qualitative analysis? What are the similarities and differences? I'm waiting for your opinion.

At the beginning, I would like to note that the subject of psychology is very complex and for the most profound understanding of it, it is necessary, first of all, to decide what lies in the foundation.

PSYCHOLOGY is a science that studies the patterns of emergence, development, and functioning of the human psyche, as well as a group of people. After we have determined what the science of psychology studies, we can move on to consider this issue more specifically.

It is worth noting that the main concepts that we will encounter in the course of reasoning on this topic are: PSYCHOLOGY, ANALYSIS, QUANTITATIVE, QUALITATIVE, PERSONALITY. And now, after clarifying the fundamental concepts, we can proceed to a specific consideration of your question.

First, let's look at what the term "ANALYSIS" means? Analysis is a research method characterized by the selection and study of individual parts of the objects of study. After we have determined what is commonly called and considered as analysis. Let's take a closer look at your question. What is quantitative analysis? What are its main features? Quantitative Analysis is a set of procedures, methods for describing and transforming research data based on the use of a mathematical and static apparatus. It is worth noting that this analysis implies the possibility of treating the results as numbers - applying methods of certain calculations. Now let's look more specifically, what is qualitative analysis? Qualitative analysis h is a set of procedures and methods for describing research data based on theoretical conclusions and generalizations, individual experience, intuition, methods of inference. In the course of this analysis, the causes of the emergence of a particular psychological phenomenon are revealed, its essential properties are revealed, development trends are established, and the contradictions of functioning are determined.

It can be added that each of these analyzes plays a certain role in psychology and under some circumstances each has its own advantages. This concludes our lesson. I believe that you have learned what properties the imagination has in psychology. If something remains incomprehensible from this topic, you can always ask your question on our website.
We wish you good luck and success in your work!

Analytical chemistry deals with the study experimental methods determining the composition of substances. Determining the composition of substances includes identifying the nature of the components that make up the substance under study, and establishing the quantitative ratios of these components.

First, the qualitative composition of the object under study is established, i.e. solve the question of what it consists of, and then proceed to determine the quantitative composition, i.e. find out in what quantitative ratios the found components are in the object of study.

Qualitative Analysis substances can be carried out by chemical, physical, physico-chemical methods.

Chemical methods of analysis are based on the use of characteristic chemical reactions to determine the composition of the analyte.

Chemical analysis of a substance is carried out in two ways: "dry way" or "wet way". Dry analysis- This chemical reactions occurring with substances during incandescence, fusion and coloring of the flame.

Wet analysis are chemical reactions that take place in electrolyte solutions. The analyte is pre-dissolved in water or other solvents. Depending on the mass or volume of the substance taken for analysis, macro-, semi-micro- and micro methods are distinguished from the technique used.

macromethod. For analysis, take 1-2 ml of a solution containing at least 0.1 g of the substance, and add at least 1 ml of the reagent solution. The reactions are carried out in a test tube, the precipitate is separated by filtration. The precipitate on the filter is washed from impurities.

Semi-micromethod. For analysis, 10-20 times less substance is taken (up to 0.01 g). Since this method works with small amounts of a substance, microtest tubes, watch or glass slides are used. Centrifugation is used to separate the precipitate from the solution.

Micromethod. When performing an analysis by this method, one or two drops of the solution are taken, and the dry matter is within 0.001 g. Typical reactions are carried out on a watch glass or porcelain plate.

When carrying out the analysis, the following operations are used: heating and evaporation, precipitation, centrifugation, checking the completeness of precipitation, separation of the solution (centrifuge) from the precipitate, washing and dissolution of the precipitate.

Heating solutions can be carried out directly with a gas burner flame, on an asbestos mesh or a water bath. A small amount of the solution is heated to a temperature not exceeding 100 ° C in a water bath, the water in which should boil evenly.

For concentration solutions are used in a water bath. Evaporation solution to a dry residue is carried out in porcelain cups or crucibles, heating them on an asbestos grid. If the dry residue after evaporation must be calcined to remove volatile salts, then the crucible is placed on a porcelain triangle and heated with a gas burner flame.

Precipitation. The precipitation reaction is carried out in conical flasks or cylindrical test tubes. A precipitating agent is added to the test solution with a pipette. The precipitant is taken in excess. The mixture is thoroughly mixed with a glass rod and rubbed against the inner walls of the test tube, this accelerates the process of sediment formation. Precipitation is often carried out from hot solutions.

Centrifugation. The precipitate is separated from the solution by centrifugation using a manual or electric centrifuge. A test tube with solution and sediment is placed in a sleeve. The centrifuge must be loaded evenly. With rapid rotation, the centrifugal force throws sediment particles to the bottom and compacts it, and the solution (centrifugate) becomes transparent. The rotation time is from 30 s to several minutes.

Checking the completeness of sedimentation. The tube is carefully removed from the centrifuge and 1-2 drops of the precipitant are added along the wall to the clear solution. If the solution does not become cloudy, then the precipitation is complete. If the solution becomes cloudy, then a precipitant is added to the test tube, the contents are mixed, heated and centrifuged again, then the sedimentation completeness check is repeated.

Separation of the solution (centrifugate) from the sediment. After making sure that the precipitation is complete, the solution is separated from the precipitate. The solution is separated from the precipitate with a drop pipette. The pipette is closed with the index finger and carefully removed from the test tube. If the selected solution is needed for analysis, then it is transferred to a clean test tube. For complete separation, the operation is repeated several times. During centrifugation, the precipitate may settle tightly to the bottom of the tube, then the solution is separated by decantation (carefully drained).

Sediment washing. The sediment (if it is examined) must be washed well; for this, a washing liquid is added, most often distilled water. The contents are thoroughly mixed with a glass rod and centrifuged, then the washing liquid is separated. Sometimes in work this operation is repeated 2-3 times.

Dissolution of the precipitate. To dissolve the precipitate, add the solvent to the test tube, stirring with a glass rod. Often, the dissolution of the precipitate is carried out by heating in a water bath.

For determining quantitative composition substance or product are used reactions of neutralization, precipitation, oxidation - reduction, complexation. The amount of a substance can be determined by its mass or the volume of the solution spent on interaction with it, as well as by the refractive index of the solution, its electrical conductivity or color intensity, and the like.

By the amount of the substance taken for research analytical methods quantitative analysis are classified as follows: macroanalysis - 1-10 g of solid, 10-100 ml of the analyzed solution; semi-microanalysis - 0.05-0.5 solids, 1-10 ml of the analyzed solution; microanalysis - 0.001-1-10 - 4 g of solid, 0.1-1 * 10 - 4 ml of the analyzed solution. In commodity practice, gravimetric (weight) and titrimetric (volumetric) methods are often used.

Gravimetric (weight) analysis- one of the methods of quantitative analysis, which allows you to determine the composition of the analyte by measuring the mass. Mass measurement (weighing) is performed on an analytical balance with an accuracy of 0.0002 g. This method is often used in food laboratories to determine moisture, ash content, the content of individual elements or compounds. Analysis can be performed in one of the following ways.

1. Defined constituent part quantitatively (as completely as possible) isolated from the test substance and weighed. This is how the ash content of products is determined. The initial product weighed on an analytical balance is burned, the resulting ash is brought to a constant mass (calcined until the mass ceases to change) and weighed.

The ash content of the product x (%) is calculated by the formula

where B is the mass of calcined ash, g;

A - the initial sample of the product, g.

2. The analyzed component is completely removed from the sample of the initial substance and the residue is weighed. This is how the moisture content of the products is determined, while the sample of the starting material is dried in an oven to constant weight.

Product moisture x (%) is calculated by the formula

where A is the initial sample of the product, g;

B is the weight of the sample after drying, g.

Volumetric Analysis- a method of quantitative analysis, where the desired substance is determined by the volume of a reagent with a precisely known concentration, spent on the reaction with this substance.

When determining by the volumetric method, a reagent with a precisely known concentration is added in small portions (dropwise) to a known volume of a solution of the analyte until its amount is equivalent to the amount of the analyte. A reagent solution with a precisely known concentration is called a titrated, working, or standard solution.

The process of slowly adding a titrated solution to a solution of an analyte is called titration. The moment when the amount of the titrated solution is equivalent to the amount of the analyte is called the equivalence point or the theoretical end point of the titration. To determine the equivalence point, indicators are used that undergo visible changes near it, expressed in a change in the color of the solution, the appearance of turbidity or precipitation.

The most important conditions for the correct conduct of volumetric analytical determinations: 1) the ability to accurately measure the volumes of solutions; 2) availability of standard solutions with precisely known concentration; 3) opportunity exact definition the end of the reaction (correct choice of indicator).

Depending on what reaction the definition is based on, the following types of volumetric method are distinguished:

method of neutralization

· oxidation-reduction method

the method of precipitation and complexation.

At the core neutralization method lies the reaction of interaction between H + and OH - ions. The method is used to determine acids, bases and salts (which react with acids or bases) in solution. For the determination of acids, titrated solutions of alkalis KOH or NaOH are used, for the determination of bases, solutions of acids HC1, H 2 SO 4 are used.

To determine the content, for example, of an acid in a solution, a volume of an acid solution accurately measured with a pipette in the presence of an indicator is titrated with an alkali solution of a precisely known concentration. The equivalence point is determined by the change in the color of the indicator. According to the volume of alkali used for titration, the acid content in the solution is calculated.

Method oxidation - reduction is based on the redox reactions that occur between the standard solution and the analyte. If the standard solution contains an oxidizing agent (reducing agent), then the analyte must contain a corresponding reducing agent (oxidizing agent). The redox method is subdivided, depending on the standard solution used, into the permanganatometry method, the iodometry method, etc.

At the heart of the method deposition are reactions accompanied by precipitation. In contrast to the gravimetric method, the sediment is not processed here, the mass of the test substance is determined by the volume of the reagent consumed in the precipitation reaction.

Every living organism, including bacteria and viruses, has unique genes that are included in the structure of DNA or RNA in a certain sequence. During a PCR study, genetic material is repeatedly copied under the influence of DNA polymerase and special temperature cycles.

There are two main polymerase chain reaction methods:

1. The classical method is the isolation of the genetic material of the pathogen by electrophoresis;
2. PCR in real time.

The methodology consists of three main steps:

• Sample preparation;
• DNA amplification;
• Detection (identification) of the genetic material of the alleged pathogen.

To conduct the study, the PCR laboratory must be divided into 3 zones, each stage of the reaction is carried out strictly in the room intended for it. Each zone must be provided with the necessary equipment, dispensers, consumables, protective clothing, used only in this room.

After registration and labeling of samples, in the sample preparation room, DNA or RNA of the pathogen is isolated from the test material by exposure to a certain temperature and special reagents. Then the process of amplification begins - the creation of numerous copies of a unique DNA fragment. It consists of 3 main steps:

• DNA denaturation - under the influence of high temperature (95 degrees), the DNA double helix unwinds into 2 chains.
• Primer annealing - special synthetic compounds (primers) are attached to the ends of DNA chains, identical to the genetic information at the ends of the desired nucleic acid fragments. The temperature required for attaching the primer is individual for a particular case, it ranges from 50 to 65 degrees C.
• With the help of the DNA polymerase enzyme at 70 - 72 degrees, a similar DNA section (amplicon) is completed between the two primers. As a "building material" special substances are used that are added to the test tube.

Amplification cycles are repeated several times, therefore, the isolated DNA is copied many times, which simplifies the process of its identification. Identification can be carried out visually after the electrophoresis of amplification products in agarose gel, or automatically using the real-time technique.

In the study by the PCR method in the "real time" mode, amplification and detection occur simultaneously in special devices. This method is the most preferable, since the study is carried out in closed test tubes, the risk of contamination and, consequently, the issuance of false positive results is reduced.

Pros and cons of the method

• The study takes only a few hours, in contrast to the long classical microbiological methods;
• High specificity from 95% to 100%, because the desired DNA fragment is unique for each specific microorganism;
• High sensitivity of the method, it is possible to detect the pathogen, even if it is represented by only one cell in the test sample;
• The pathogen can be identified both qualitatively and quantitative method. This is very important when isolating opportunistic microorganisms that do not cause disease in small quantities;
• The ability to determine the genotype of the pathogen (hepatitis C, HIV infection). This is necessary for rational treatment and prognosis of possible complications;
• The ability to identify a genetic predisposition to the disease, thereby preventing its development;
• It is possible to isolate almost any source of infection, and modern techniques also make it possible to identify the total microflora in the test sample, for example, the vaginal biocenosis.

• The possibility of obtaining both a false positive and a false negative sample in case of non-compliance with the rules for sampling, errors during the study;
• High cost of analysis.

Application

Almost any sample can be examined by PCR (blood, urine, cerebrospinal fluid, scrapings from the cervical canal and urethra, hair follicles, sperm, etc.). This technique is widely used for the diagnosis of STDs (gonorrhea, chlamydia, ureaplasmosis, mycoplasmosis, trichomoniasis). It can be used to identify pathogens of tuberculosis, diphtheria, pneumonia, viral hepatitis, HIV infection, toxoplasmosis, cytomegalovirus and herpes infections, salmonellosis, etc.

A polymerase chain reaction is used to establish paternity by comparing the DNA of the parent and child, identifying genetic abnormalities and the body's hereditary predisposition to various diseases.

Preparation for the delivery of the analysis

• It is recommended to donate blood strictly on an empty stomach.
• Before taking a smear from the urethra or cervical canal, you should refrain from sexual activity for three days, it is necessary to take an analysis no earlier than a month after the end of the course of antibiotic therapy, otherwise the result may be false positive. The PCR method detects the DNA of even a dead pathogen, so it is better to conduct a study after a complete cell renewal.
• Urine should be collected in a sterile container.

The answer will most often be ready in a couple of days, depending on the capabilities of the laboratory.

Deciphering the results

When using a qualitative methodology, there can be only 2 possible answers: positive or negative. A positive result indicates the presence of an isolated microorganism in the sample, a negative result indicates the absence.

The quantitative result should be evaluated by the attending physician, applied individual approach in each specific case. The specialist, taking into account the received answer, decides on the need for treatment, dosage of drugs, specifies the form and stage of the disease.

When determining the genetic profile (predisposition to thrombophilia, breast cancer), after deciphering the result, the doctor can assess the degree of risk of developing the disease, as well as prescribe a special diet and preventive measures.

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Polymerase chain reaction - information for patients

Polymerase chain reaction (PCR) is a complex laboratory method widely used in medicine and other branches of science. At one time, PCR diagnostics became a big breakthrough in science. It can be said that this is one of major discoveries 20th century in medicine. For the discovery of the method, Keri Mullis, a biochemist, received the Nobel Prize in 1993.

For a long time, infections have collected a bitter tribute from mankind. The plague alone claimed hundreds of thousands of lives in the Middle Ages. In the successful fight against epidemics, an important point is accurate and timely diagnosis.

PCR studies are usually carried out in a laboratory at the clinic. Despite the fact that the PCR test for infection is quite expensive, the high accuracy compensates for the price. To establish an accurate diagnosis, it is enough to do an analysis once. Other methods may require additional or repeat tests.

How is an infectious disease test done?

Most often, serological and cultural methods are used to diagnose infections. In the first case, antibodies to the infectious agent in the blood serum are determined. In the second case, the biological material obtained from a sick person is used to sow a special environment favorable for the growth of pathogen colonies. In both cases, diagnosis can take days or even weeks.

PCR examination can be carried out with any biological materials obtained from a sick person. Blood and other biological, physiological and pathological fluids and media can serve as samples. You can do PCR of urine or feces.

Most often, viral and atypical infections are determined by PCR, since they may not be amenable to conventional diagnosis due to the characteristics of the pathological process they cause. In order to diagnose these infections, it takes time for the body to start producing antibodies, which are determined by serological methods. However, in some cases this is unacceptable.

With the help of PCR, the human immunodeficiency virus can be determined in days or weeks, as accurately as possible, without the period of the seronegative window characteristic of other methods. (The seronegative window is the interval from the moment of infection in which the body has not yet begun to produce a sufficient amount of antibodies to determine).

The PCR method - in vitro means the laboratory determination of infection in samples isolated from the patient.

To carry out the polymerase chain reaction, a set of special reagents is required.

The test material is added to the test tubes with reagents. The tubes are placed in special device- PCR amplifier. It serves to amplify (increase the number) of the desired DNA or RNA fragments. The PCR cycler runs in a cyclic mode. Each cycle, if the DNA or RNA sequence of the pathogen is present in the samples, copies of fragments of these nucleic acids accumulate in the solution. It is possible to determine both the presence of the pathogen and its amount in the samples.

Types of PCR

PCR analysis - qualitative gives the following result:

• PCR - negative, the desired pathogen was not found in the samples;
• PCR - positive, sequences characteristic of a particular pathogen were found in the samples.

When the PCR result is positive, it indicates with 95% accuracy the presence of a diagnosed infection. The accuracy of PCR kits used for diagnostics reaches 100%.

5% of erroneous results usually depend on the human factor. Thus, violations of the rules for storing reagents and research techniques can significantly reduce the accuracy of analyzes.

Quantitative PCR analysis defines such a thing as viral load. At the same time, it is possible to determine how many sets of DNA of the pathogen were contained in the samples obtained from the patient. The more, the more severe the infection. You can also determine the success of treatment by reducing the viral load.

Delivery of biomaterial for PCR

Delivery of PCR tests is carried out in the clinic, usually in the morning. During the visit to the doctor, you will be told what to donate: blood, urine, smear or scraping. PCR is able to detect pathogens regardless of the degree of contamination of the material.

In theory, the presence of only one pathogen in the samples is sufficient for a positive analysis. In practice, they try to create more favorable conditions. There are some rules for this:

• if you are taking a swab or scraping from the genitals, you should refrain from sexual intercourse 3 days before the test;
• do not wash or douche with antibacterial agents on the eve of the test;
• 3 hours before taking a smear from the urethra, you should be patient and not urinate.

In the case when the patient donates blood, you can not adhere to these rules.

Research results

PCR results are usually ready within a day after the test. Qualitative analysis looks simple. PCR decoding is not required, since usually the pathogen is indicated in the first column, and the result in the second. For example, like this:

(PCR) Ureaplasma urealiticum

(PCR) Herpes simplex

In parentheses indicates the method - PCR. It's not hard to interpret. In the patient from the example, cytomegalovirus (CMV) and herpes were detected by a qualitative PCR method. Ureaplasma and chlamydia: pathogens were not found.

Quantitative analysis gives a numerical result, usually in IU / ml. This means that in 1 ml of the test sample, a certain number of copies of DNA or RNA of the pathogen, in international units, was found. Depending on the size, the severity of the infection is determined. Usually, blood is examined to determine the viral load, since viruses circulate freely in the blood during the disease.

Where can I do PCR

It is important to take tests in a clinic that has proven itself well. Although the method is very accurate, its results are affected by adherence to the study protocol. You should not look for a clinic, guided by the results of queries in a search engine, such as: PCR Moscow, where to do it or PCR Stavropol clinic. As a rule, in which place it is better to do a PCR analysis, your doctor recommends.

If the PCR result is positive, it is necessary to do a second study in another laboratory. This will eliminate the error depending on the human factor.

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Additions have been made to the IVF program for compulsory medical insurance in 2018.

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Diagnosis of hepatitis C by PCR study

Polymerase chain reaction (PCR) is increasingly being used in clinical practice to determine the causes of various viral diseases, including the diagnosis of viral hepatitis C.

Advice from hepatologists

In 2012, there was a breakthrough in the treatment of hepatitis C. New direct-acting antiviral drugs were developed that have a 97% chance of completely curing you of the disease. Since then, hepatitis C has been officially considered a completely curable disease in the medical community. In the Russian Federation and CIS countries, drugs are represented by the brands sofosbuvir, daclatasvir and ledipasvir. At the moment, there are a lot of fakes on the market. Medicines of good quality can only be purchased from companies that have licenses and relevant documentation.

For its diagnosis, PCR in its various modifications is actively used. Using PCR for hepatitis C, it is possible to establish the presence of hepatitis C virus RNA in the patient's blood and make an accurate diagnosis.

Analysis Variations

The PCR technique became available to doctors several decades ago. It is based on multiple copying of a certain fragment of viral or bacterial RNA or DNA, followed by detection (recognition of this fragment) in the patient's blood serum.

At the same time, there are two fundamentally different methods of PCR research: quantitative and qualitative.

A qualitative method only allows answering the question: is there in the biological material (blood serum, saliva, seminal fluid, etc.) the genetic material of a certain virus?

The quantitative method, in turn, allows you to determine the amount of this genetic material, which is necessary in some cases to determine the stage of the disease or evaluate the effectiveness of therapy.

A qualitative version of PCR for the disease

The use of a qualitative variant of PCR analysis in this disease makes it possible to detect the presence of viral hepatitis C RNA in the biological fluids (blood serum, saliva, etc.) of the patient. In this case, the result of the analysis can only be of two types: positive or negative. In this case, its correct decoding is very important.

• a positive result in determining the RNA of viral hepatitis C tells the doctor that the studied biological fluid contains RNA of this virus. Accordingly, the patient is infected with it, which means that a diagnosis of viral hepatitis C is possible. However, it is always worth remembering the possibility of false positive test results;
• a negative result of the PCR analysis indicates the absence of hepatitis C virus RNA in the biological fluid under study, or the content of RNA molecules in the test fluid was too low and was below the sensitivity limit of the PCR method. A negative test result may not always indicate the absence of the virus in the blood. The possibility of false negative test results should always be considered by the treating physician.

With the development of an acute form of hepatitis C disease, a qualitative PCR study makes it possible to establish the fact of the disease already 1-3 weeks after the virus enters the human body.

False negative results may result from:

• getting into the biological material (blood) of polluting substances;
• the use of Heparin to prevent blood clotting in vitro or its use by the patient;
• penetration into the test material of substances from environment blocking enzymes used in PCR.

Quantitative PCR variant

The use of quantitative PCR analysis allows you to determine not only the very fact of the presence of the virus in the blood, but also the number of viral particles in any biological fluid (the so-called viral load). Using this type of PCR, you can determine the number of copies of hepatitis C virus RNA that circulate in a certain volume.

The result of this type of PCR is expressed as numerical values, where the unit of measurement is international units per milliliter - IU / ml.

Carrying out a similar type of PCR diagnostics is used on certain days of treatment of viral hepatitis C. The first determination of the viral load occurs when a sick person enters the hospital. In the future, the analysis is carried out on the 1st, 4th, 12th and 24th weeks from the start of application medicines. Already at the 12th week, you can tell whether the therapy is effective or not.

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Special preparation of the patient for the study is not required. It is recommended not to smoke on the day of the test. Blood from a vein is used as the test material.

After quantitative PCR has been carried out, it is necessary to decipher the results obtained. The concept of "norm" in such cases does not exist. For decoding, a specially developed gradation of indicators is used:

• result of the study: not detected - RNA of viral hepatitis C was not detected in the patient's venous blood (the result is negative), or it is contained in a very low amount that does not allow the method to determine it (<40 ME/мл – порог чувствительности количественного ПЦР);
• research result:<8*10 5 МE/мл – положительный результат теста. Такой уровень вирусной нагрузки очень низкий. Является показателем эффективности терапии и благополучного течения заболевания;
• test result: >8*10 5 IU/ml – positive test result. The load level is very high. Poor prognosis of the course of the disease and the need to correct or replace the drugs used.

It is important to remember that the obtained level of viral load does not reflect the severity of the pathology and the degree of destruction of the liver. For this, there are other methods of biochemical research. For the correct selection of treatment methods, it is necessary to know the genotype of the hepatitis C virus.

1. A high concentration of viral particles in biological fluids, and especially in the blood, is associated with a high risk of transmission of the virus during sexual intercourse or during pregnancy from mother to fetus.
2. The number of viral particles is a reflection of the effectiveness of the drugs used and allows you to rationally choose the drugs and doses used.

Ultrasensitive method of PCR diagnostics

To date, you can go through the so-called ultra PCR to determine the hepatitis C virus. This method is completely called PCR with real-time hybridization-fluorescence research.

When ultra PCR is indicated:

1. In cases of suspected viral hepatitis C in patients with latent forms of the disease.
2. In cases where the patient has antibodies to the hepatitis C virus, but not confirmed by PCR diagnostics.
3. To evaluate the effectiveness of the treatment and confirm the fact of recovery.
4. As a screening technique for early detection of a disease in people in a population.

For the study, as a rule, the patient's venous blood is used. The sensitivity of the ultra method is less than 10 IU/l, which is several times higher than that of standard quantitative and qualitative PCR diagnostics. The appointment of ultra PCR is carried out by an infectious disease specialist or a hepatologist.

The decisive step in making a diagnosis and evaluating treatment is the correct interpretation of the results obtained with the ultra PCR method. It is always worth remembering that there is a small chance of getting false negatives and false positives.

To eliminate such situations, it is necessary to exclude contamination of blood samples and laboratory materials. With the use of ultra PCR, it is possible to avoid situations that lead to false negative results, and thus complicate the diagnosis.

Judging by the fact that you are now reading these lines, victory in the fight against Hepatitis C is not on your side yet.

And have you already taken toxic drugs that had a bunch of side effects? It is understandable, because ignoring the disease can lead to serious consequences. Fatigue, weight loss, nausea and vomiting, yellowish or grayish skin tone, bitter taste in the mouth, body and joint aches. Are all these symptoms familiar to you?

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What is the difference between qualitative and quantitative PCR?

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Please tell me which PCR test for STD smears is more informative: qualitative or semi-quantitative? How do they differ from each other?

In general, there are 2 types of PCR - qualitative (yes / no) and quantitative. Quantitative requires other equipment, much more expensive, used mainly for HIV and hepatitis.

Semi-quantitative analysis is, in a certain sense, an inadequate substitute for quantitative analysis; it is not necessary to do it:

This is not quantitative analysis.

It is usually more expensive

In the diagnosis of STDs, the number of microorganism does not matter.

Usually this is a separate study.

Special media are used, usually imported, more often MYCOPLASMA DUO + ​​antibiogram SIR (BIORAD, France) or MYCOPLASMA IST (BioMerier), but it is more expensive. The cost of determining the order of $ 10 for both infections.

It makes sense to do PCR only in the sense of diagnosing mycoplasmas that are not determined by sowing - M.genitalium.

It is also possible as a cheap way to identify all mycoplasmas in general - usually called Mycoplasma spp. (i.e. all species of the genus Mycoplasma). However, non-pathogenic ones are also determined, so a negative answer is of great value.

What is the name of the diagnosis if one elementary or reticulum body from the main forms of chlamydia is found? Similarly, the question is about the detection of one or more pairs of gonococci, as well as one Trichomonas cell. Thank you for your attention and the discussion that has begun. Sincerely, Vladimir.

How are you going to find ONE elemental or reticular body?

With light microscopy, this is impossible; with immunofluorescence, there are criteria for issuing a response - usually 5-10 objects with a characteristic glow, depending on the set.

With PCR and ELISA for antigens, the question is generally inadequate.

The sensitivity of most Russian PCR kits is about 1000 genocopies per ml of sample.

The answer is similar - but how to do it?

Regarding trichomonas, options are possible, but still ONE cell is casuistry, and you can always check the result by another method, the same PCR, by the way.

This is impossible for gonorrhea - according to a Gram-stained smear, a diagnosis of gonorrhea can only be made in acute gonorrhea in men (and in America this is also only a presumptive diagnosis), and one pair of gonococci can be found VERY RARE. All other cases are "gram-negative intracellular diplococci".

When sowing, you get a Goncoccus COLONY, which you must identify (now this is not a problem).

For PCR, see above.

Gonococcus is determined by microscopy of Gram-stained smears;

Trichomonas by microscopy of a native smear;

Chlamydia - PCR or seeding on special media;

Mycoplasma - sowing on special media

So? It's enough? Does antibody levels play a role in the diagnosis of STDs?

Bakposev with subsequent identification of colonies with modern kits for the differentiation of Neisseria. Unfortunately, it's rarely done. In KVD, as a rule, identification on sugars is not carried out, although it should.

Bacterioscopy (acute gonorrhea in men)

Microscopy of the native preparation and its modifications

Sowing on Trichomonas

Microscopy of stained smears (set only if typical forms are found, and not "scraps of Trichomonas")

Sowing on a cell. culture or embryos (difficult)

It is only possible to suggest a benefit in ascending chlamydial infection or Reiter's syndrome.

Often in the CIS they lead to multiple treatment "until the titer disappears"

As a method of control of cure - categorically not!

I think STD screening will switch to nucleic acid amplification methods (the same PCR)

For chlamydia, this is already the case, gonorrhea - more and more so.

Compared to a bacterial or virological study, PCR is simpler and faster for both the clinician and the laboratory, and therefore more reliable.

Backstudy will remain the reference method. This is my prediction.

Do you think there is at least one adult person on Earth who more than once, during his life, “faced” with one or another subspecies of chlamydia? How, however, with most of the other potentially pathogenic microflora? In the vast majority of cases, such meetings (usually in low credits) end tragically for this microflora. 🙂 Much less often a carrier (usually temporary), even less often a disease.

And the second question, what do you think: why, for many millennia of coexistence of a person with at least the same Chlamydia trachomatis, when from chlamydia (this is not a reservation, because it is often treated for a bac. agent, which was detected using PCR 😎) only they didn’t treat, but, in general, their coexistence was not known, all without exception did not get chlamydia? Indeed, in the history of mankind there were quite a few periods when polygamous sexual relations were the norm.

From here often and "persistence". Venereologists refuse to believe the results - "find nothing, here we have smears." etc.

But let's not forget the question of the respected ksena: "Good afternoon!

Please tell me which PCR test for STD smears is more informative: qualitative or semi-quantitative? How are they different from each other?"

This question, in my opinion, is from the area of ​​​​interest of human cognition. And knowledge has no boundaries. Over time, the question of pathological molecules (prion proteins), then the tension in torsion fields at the level of atoms, etc., will be interesting, and then attention will switch to the macrocosm. There will be questions from astrology about the influence of planets on our health. His Majesty EXPERIENCE. But many thanks for this question. All the best, with respect to all those present, Vladimir.

But let's not forget the question of the respected ksena: "Good afternoon!

Please tell me which PCR test for STD smears is more informative: qualitative or semi-quantitative? How are they different from each other?"

The analysis of a substance can be carried out in order to establish its qualitative or quantitative composition. Accordingly, a distinction is made between qualitative and quantitative analysis.

Qualitative analysis allows you to establish what chemical elements the analyzed substance consists of and what ions, groups of atoms or molecules are included in its composition. When studying the composition of an unknown substance, a qualitative analysis always precedes a quantitative one, since the choice of a method for the quantitative determination of the constituent parts of the analyzed substance depends on the data obtained during its qualitative analysis.

Qualitative chemical analysis is mostly based on the transformation of the analyte into some new compound with characteristic properties: color, a certain physical state, crystalline or amorphous structure, a specific smell, etc. The chemical transformation that occurs in this case is called a qualitative analytical reaction, and the substances that cause this transformation are called reagents (reagents).

When analyzing a mixture of several substances with similar chemical properties, they are first separated and only then characteristic reactions are carried out for individual substances (or ions), therefore, qualitative analysis covers not only individual reactions for detecting ions, but also methods for their separation.

Quantitative analysis allows you to establish the quantitative ratio of the parts of a given compound or mixture of substances. Unlike qualitative analysis, quantitative analysis makes it possible to determine the content of individual components of the analyte or the total content of the analyte in the test product.

Methods of qualitative and quantitative analysis, allowing to determine the content of individual elements in the analyzed substance, are called elements of analysis; functional groups - functional analysis; individual chemical compounds characterized by a certain molecular weight - molecular analysis.

A set of various chemical, physical and physico-chemical methods for separating and determining individual structural (phase) components of heterogeneous systems that differ in properties and physical structure and are limited from each other by interfaces is called phase analysis.

Qualitative analysis methods

Qualitative analysis uses characteristic chemical or physical properties of the substance to establish the composition of the substance under investigation. There is absolutely no need to isolate the discovered elements in their pure form in order to detect their presence in the analyzed substance. However, the isolation of metals, non-metals, and their compounds in pure form is sometimes used in qualitative analysis for their identification, although this way of analysis is very difficult. To detect individual elements, simpler and more convenient methods of analysis are used, based on chemical reactions characteristic of the ions of these elements and occurring under strictly defined conditions.

An analytical sign of the presence of the desired element in the analyzed compound is the release of a gas that has a specific odor; in the other - the precipitation, characterized by a certain color.

Reactions between solids and gases. Analytical reactions can take place not only in solutions, but also between solid and gaseous substances.

An example of a reaction between solids is the reaction of the release of metallic mercury when dry salts of it are heated with sodium carbonate. The formation of white smoke from the interaction of gaseous ammonia with hydrogen chloride can serve as an example of an analytical reaction involving gaseous substances.

The reactions used in qualitative analysis can be divided into the following groups.

1. Precipitation reactions, accompanied by the formation of precipitates of various colors. For example:

CaC2O4 - white

Fe43 - blue,

CuS - brown - yellow

HgI2 - red

MnS - flesh - pink

PbI2 - golden

The resulting precipitates may differ in a certain crystal structure, solubility in acids, alkalis, ammonia, etc.

2. Reactions accompanied by the formation of gases with a known odor, solubility, etc.

3. Reactions accompanied by the formation of weak electrolytes. Among such reactions, which result in the formation of: CH3COOH, H2F2, NH4OH, HgCl2, Hg(CN)2, Fe(SCN)3, etc. Reactions of the same type can be considered reactions of acid-base interaction, accompanied by the formation of neutral water molecules, reactions of the formation of gases and precipitates that are poorly soluble in water, and complexation reactions.

4. Reactions of acid-base interaction, accompanied by the transition of protons.

5. Complexation reactions accompanied by the addition of various legends - ions and molecules - to the atoms of the complexing agent.

6. Complexation reactions associated with acid-base interaction

7. Oxidation reactions - reductions, accompanied by the transition of electrons.

8. Oxidation reactions - reductions associated with acid - base interaction.

9. Oxidation-reduction reactions associated with complex formation.

10. Oxidation reactions - reductions, accompanied by the formation of precipitation.

11. Ion exchange reactions occurring on cation exchangers or anion exchangers.

12. Catalytic reactions used in kinetic methods of analysis

Wet and dry analysis

The reactions used in qualitative chemical analysis are most often carried out in solutions. The analyte is first dissolved and then the resulting solution is treated with appropriate reagents.

To dissolve the analyte, distilled water, acetic and mineral acids, aqua regia, aqueous ammonia, organic solvents, etc. are used. The purity of the solvents used is an important condition for obtaining correct results.

The substance transferred into solution is subjected to systematic chemical analysis. A systematic analysis consists of a series of preliminary tests and sequentially performed reactions.

The chemical analysis of test substances in solutions is called wet analysis.

In some cases, substances are analyzed dry, without transferring them into solution. Most often, such an analysis comes down to testing the ability of a substance to color a colorless burner flame in a characteristic color or to impart a certain color to a melt (the so-called pearl) obtained by heating a substance with sodium tetraborate (borax) or sodium phosphate ("phosphorus salt") in a platinum wire.

Chemical and physical method of qualitative analysis.

Chemical methods of analysis. Methods for determining the composition of substances based on the use of their chemical properties are called chemical methods of analysis.

Chemical methods of analysis are widely used in practice. However, they have a number of disadvantages. So, to determine the composition of a given substance, it is sometimes necessary to first separate the component to be determined from foreign impurities and isolate it in its pure form. The isolation of substances in pure form is often a very difficult and sometimes impossible task. In addition, in order to determine small amounts of impurities (less than 10-4%) contained in the analyte, it is sometimes necessary to take large samples.

Physical methods of analysis. The presence of a particular chemical element in a sample can be detected without resorting to chemical reactions, based directly on the study of the physical properties of the substance under study, for example, coloring a colorless burner flame in characteristic colors with volatile compounds of certain chemical elements.

Methods of analysis, by which it is possible to determine the composition of the substance under study, without resorting to the use of chemical reactions, are called physical methods of analysis. Physical methods of analysis include methods based on the study of optical, electrical, magnetic, thermal and other physical properties of the analyzed substances.

Among the most widely used physical methods of analysis are the following.

Spectral qualitative analysis. Spectral analysis is based on the observation of emission spectra (emission spectra, or radiation) of the elements that make up the analyte.

Luminescent (fluorescent) qualitative analysis. Luminescent analysis is based on the observation of luminescence (light emission) of analytes caused by the action of ultraviolet rays. The method is used to analyze natural organic compounds, minerals, medicines, a number of elements, etc.

To excite the luminescence, the test substance or its solution is irradiated with ultraviolet rays. In this case, the atoms of matter, having absorbed a certain amount of energy, pass into an excited state. This state is characterized by a larger supply of energy than the normal state of matter. During the transition of a substance from an excited to a normal state, luminescence occurs due to excess energy.

Luminescence that decays very quickly after cessation of irradiation is called fluorescence.

Observing the nature of the luminescent glow and measuring the intensity or brightness of the luminescence of a compound or its solutions, one can judge the composition of the substance under study.

In some cases, the definitions are based on the study of fluorescence resulting from the interaction of the analyte with certain reagents. Fluorescent indicators are also known, which are used to determine the reaction of the medium by changing the fluorescence of the solution. Luminescent indicators are used in the study of colored media.

X-ray diffraction analysis. With the help of X-rays, it is possible to determine the size of atoms (or ions) and their mutual arrangement in the molecules of the sample under study, i.e., it is possible to determine the structure of the crystal lattice, the composition of the substance, and sometimes the presence of impurities in it. The method does not require chemical treatment of the substance and its large quantities.

Mass spectrometric analysis. The method is based on the determination of individual ionized particles deflected by an electromagnetic field to a greater or lesser extent depending on the ratio of their mass to charge (for more details, see Book 2).

Physical methods of analysis, having a number of advantages over chemical ones, in some cases make it possible to solve problems that cannot be resolved by methods of chemical analysis; using physical methods, it is possible to separate elements that are difficult to separate by chemical methods, as well as to conduct continuous and automatic recording of readings. Very often, physical methods of analysis are used along with chemical ones, which makes it possible to use the advantages of both methods. The combination of methods is of particular importance when determining negligible amounts (traces) of impurities in the analyzed objects.

Macro, semi-micro and micro methods

Analysis of large and small quantities of the test substance. In the old days, chemists used large quantities of the substance to be analyzed. In order to determine the composition of a substance, samples of several tens of grams were taken and dissolved in a large volume of liquid. This also required chemical glassware of the appropriate capacity.

At present, chemists manage in analytical practice with small amounts of substances. Depending on the amount of the analyte, the volume of solutions used for analysis, and mainly on the technique used to perform the experiment, analysis methods are divided into macro-, semi-micro- and micro-methods.

When performing a macro analysis, a few milliliters of a solution containing at least 0.1 g of the substance is taken to carry out the reaction, and at least 1 ml of the reagent solution is added to the test solution. The reactions are carried out in test tubes. During precipitation, voluminous precipitates are obtained, which are separated by filtration through funnels with paper filters.

Drop analysis

Technique for carrying out reactions in drop analysis. The so-called drop analysis, introduced into analytical practice by N. A. Tananaev, has acquired great importance in analytical chemistry.

When working with this method, the phenomena of capillarity and adsorption are of great importance, with the help of which it is possible to open and separate various ions in their joint presence. In drop analysis, individual reactions are carried out on porcelain or glass plates or on filter paper. In this case, a drop of the test solution and a drop of a reagent that causes a characteristic coloration or the formation of crystals are applied to the plate or paper.

When performing the reaction on filter paper, the capillary-adsorption properties of the paper are used. The liquid is absorbed by the paper, and the resulting colored compound is adsorbed on a small area of ​​the paper, thereby increasing the sensitivity of the reaction.

Microcrystalloscopic analysis

The microcrystalloscopic method of analysis is based on the detection of cations and anions by means of a reaction, as a result of which a compound is formed that has a characteristic crystal shape.

Previously, this method was used in qualitative microchemical analysis. Currently, it is also used in drip analysis.

To examine the resulting crystals in microcrystalloscopic analysis, a microscope is used.

Crystals of a characteristic shape are used when working with pure substances by introducing a drop of a solution or a crystal of a reagent into a drop of the test substance placed on a glass slide. After a while, clearly distinguishable crystals of a certain shape and color appear.

Powder grinding method

To detect some elements, the method of grinding a powdered analyte with a solid reagent in a porcelain plate is sometimes used. The element to be discovered is detected by the formation of characteristic compounds that differ in color or odor.

Methods of analysis based on heating and fusion of a substance

pyrochemical analysis. For the analysis of substances, methods based on heating the test solid or its fusion with appropriate reagents are also used. Some substances, when heated, melt at a certain temperature, others sublime, and precipitation characteristic of each substance appears on the cold walls of the device; some compounds, when heated, decompose with the release of gaseous products, etc.

When the analyte is heated in a mixture with the appropriate reagents, reactions occur, accompanied by a change in color, the release of gaseous products, and the formation of metals.

Spectral qualitative analysis

In addition to the above-described method of observing with the naked eye the coloring of a colorless flame when a platinum wire with an analyte is introduced into it, other methods of studying light emitted by incandescent vapors or gases are currently widely used. These methods are based on the use of special optical devices, the description of which is given in the physics course. In such spectral devices, the decomposition into a spectrum of light with different wavelengths occurs, emitted by a sample of a substance heated in a flame.

Depending on the method of observing the spectrum, spectral instruments are called spectroscopes, which are used to visually observe the spectrum, or spectrographs, in which spectra are photographed.

Chromatographic analysis method

The method is based on the selective absorption (adsorption) of individual components of the analyzed mixture by various adsorbents. Adsorbents are called solids, on the surface of which the adsorbed substance is absorbed.

The essence of the chromatographic method of analysis is briefly as follows. A solution of a mixture of substances to be separated is passed through a glass tube (adsorption column) filled with an adsorbent.

Kinetic methods of analysis

Methods of analysis based on measuring the reaction rate and using its magnitude to determine the concentration are combined under the general name of kinetic methods of analysis (K. B. Yatsimirsky).

Qualitative detection of cations and anions by kinetic methods is carried out quite quickly and relatively simply, without the use of complex instruments.

task qualitative chromatographic analysis is the interpretation of chromatograms or, in other words, the identification of peaks in a chromatogram. To do this, use the following methods.

Substance addition method is based on the sequential introduction of substances into the analyzed mixture, the presence of which is supposed to be in it. If after that one of the peaks in the chromatogram increases (the retention time coincides), then the peak of the analyzed mixture can be identified with the introduced compound. However, this condition is only necessary, but not sufficient for identification: several substances can have the same (or very close) retention time, and not one. For the reliability of the analysis, such studies are carried out using columns with stationary phases of different nature (polar and non-polar).

Comparison method with tabular data involves determining the qualitative composition of the analyzed mixture by comparing the experimentally determined relative retention volumes of substances (under normal analysis conditions with respect to standard substances) with similar tabular values. To improve the reliability of chromatographic identification, the analysis is carried out using data obtained with phases that are different in nature.

Calculation Methods and Correlation Relations are used in cases where there is no data for the studied compounds in the tables of relative retention volumes. Correlations are used between the logarithm of the retention values ​​and the properties of the analyzed compounds (for example, the number of carbon atoms, boiling point, etc.). So, for example, for the values ​​of retained volumes of alkanes, the following equation is true:

where Г,у is the increment of the logarithm of the retention value corresponding to a certain combination of bonds (structural element); n,j- the number of structural elements of the type ij in the compound molecule. Obtained in this way V R compared with experimental values: if they are close, there is reason to believe that the identified peak corresponds to the intended connection.

Also used identification by Kovacs indices. As a result of the experiments, it was found that within the same homologous series of various classes of organic compounds (alkanes, alcohols, aldehydes, etc.) in the coordinates:

where P- the number of carbon atoms in the homologue, linear dependencies are obtained (Fig. 5.12).

These dependencies can be used for qualitative analysis of various derivatives of hydrocarbons. Thus, E. Kovacs proposed to characterize retention by the number of carbon atoms (multiplied by 100) that an n-alkane has, so that its retained volume coincides with the retained volume of the substance under study.

Rice. 5.12.

Y - line for n-alkanes;2 - line for homologues

The number of carbon atoms of an n-alkane (usually a fractional value multiplied by 100) is called Kovacs index of this substance J. The Kovacs indices for various stationary phases are well reproducible and tabulated.

the value J any connection for a given stationary phase can be determined graphically, as shown in Fig. 5.12. For this purpose, on the selected stationary phase, the dependence is obtained gV R from P for a number of n-alkanes (pentane, hexane, heptane, etc.).

The data obtained are plotted on a graph lgK fl from their 100. Next, measure UK of all substances of the mixture under study and determine them according to the schedule J, in fig. 5.12 Kovacs index Ud-equal to 598.

For members of any homologous series of alkane derivatives (carboxylic acids, aldehydes, etc.), one can obtain a linear dependence similar to that for alkanes (line 2 in fig. 5.12). The horizontal shift of these two lines relative to each other contributes to the Kovacs index of a functional group (carboxylic, carbonyl, etc.) or a multiple bond. This contribution is called homomorphic factor, its value for many compounds is determined and tabulated

The sum of these homomorphic factors, added to the number n c x 100 base alkane, makes it possible to calculate the Kovacs index for the alleged compound (according to) scientific sources and compare it with the experimental value. The proximity of these values ​​allows us to conclude that the peak on the chromatogram corresponds to the expected substance.

An important step in chromatographic analysis is quantitative interpretation of chromatograms, as a result of which the content of components in the analyzed mixture is determined. The accuracy of the results obtained depends on a number of factors, in particular, on the chosen method of analysis, the characteristics of the detector used, the method of calibration and calculation, and the nature of the analyzed components.

The amount of substance in the chromatographic zone is proportional to the area of ​​the chromatographic peak in the chromatogram. There are several methods for determining the area of ​​chromatographic peaks based on the assumption that the shape of the peak corresponds to a Gaussian curve. Most often, it is defined as the product of the height of the peak and its width at half height: see formula (5.8). Chromatographs of the latest generations are controlled by a computer; in this case, the peak area is calculated by software and displayed on the monitor screen.

The area of ​​the peak on the chromatogram depends not only on the amount of substance in the chromatographic zone, but is also determined by the characteristics of the detector and the conditions of the analysis. So, for different substances, even at their equal concentration in the analyzed mixture, peaks of unequal area are obtained on the chromatogram. Therefore, for quantitative analysis, it is not enough just to determine the area of ​​chromatographic peaks. There is a need to establish for each sample substance the coefficient of proportionality between the peak area and its content (concentration) in the analyzed mixture. In other words, the detector should be calibrated under the chosen analysis conditions. The following calibration methods are commonly used.

Absolute calibration method experimentally determine for each component of the analyzed mixture the dependence of the area of ​​the chromatographic peak on its absolute amount in the sample. This dependence is usually presented in the form of a graph or an empirical equation. Detector sensitivity can change over time, so the absolute calibration needs to be checked and adjusted periodically. When recalibrating, you can limit yourself to checking a few points on the calibration curve.

Internal standard method a substance (internal standard) with a known concentration of Cs t is introduced into the analyzed mixture. Beforehand, for each substance of the mixture, a calibration graph (or equation) is obtained that relates SfJSct with Sv/Co, where S B and 5 St - the area of ​​the peaks of the analyte and internal standard, Sv - the concentration of the analyte in the calibration mixture. During the study, the areas of the peaks of the analyzed substances and the internal standard are determined on the chromatogram, their ratio is calculated, and Sv / Q is found from the calibration graph; t. Further, according to the known Сс t, unknown concentrations of substances Sv-

The use of the internal standard method makes it possible to significantly increase the accuracy of measurements and makes periodic correction of the calibration curve unnecessary. Indeed, a change in the experimental conditions equally affects the change in the parameters of the chromatogram of the standard substance and the components of the sample.

Another advantage of the method is that it is no longer necessary to maintain the exact volume of sample fed to the column. In this case, it is also optional to separate all the peaks in the chromatogram: it is enough that the peaks of the substances of interest and the standard come out separately.

To improve the accuracy of the analysis, it is desirable that the substance used as a standard be close to the components to be determined in terms of retention and content in the analyzed mixture.

Also used calibration with correction factors. Peak area / "th component S, on the chromatogram is proportional to its amount d, in the mixture introduced into the column:

Here to,- correction factor of the substance. In the event that all substances of the analyzed mixture give separate (separated) peaks on the chromatogram, it is possible to calculate the fraction of the / "-th component by the method of internal normalization:

then summation is performed over all peaks. If the numerator and denominator of the right side of the equation are divided by the correction factor of any substance taken as a standard (? st), then we get the equation:

where k, tn \u003d k, / k„ - relative correction factor. It is easy to determine it experimentally by making mixtures of a certain composition of each substance paired with a standard one, or a mixture of all substances of a known composition, including a standard substance. After obtaining chromatograms with such a composition of substances and determining the peak areas of all components, one can find R: ota for all substances from the ratio:

where qjqci- corresponds to the ratio of the amounts of the i-th component and the standard in the initial mixture. Quantities q may be determined by mass (g) or by quantity (mol), from which mass or molar relative correction factors are calculated respectively. Accordingly, mass fractions are determined with mass coefficients, and molar fractions of substances in the mixture are determined with molar coefficients.