Of all the OM microcomponents, vitrinite is the best in terms of indicativeness in studying the degree of catagenetic transformation. The fact is that, for reliable diagnostics, a microcomponent is needed, which must have a regular change in properties during the transformation process, at the same time it must be widely distributed in the OM. Vitrinite meets all the above requirements, unlike other microcomponents of coals and DOM. Which either merge with the total organic mass of coal already at the middle stages of catagenesis (leuptinite), or weakly and unevenly react to changes in environmental parameters (fusinite). And only vitrinite changes its properties naturally gradually and is very easy to diagnose.
It is on the basis of the reflectivity of vitrinite that most of the scales for determining the degree of catagenesis are built. In addition to it, other microcomponents of DOM are also used, but to a lesser extent. The method is based on the pattern of increase in gloss during catagenesis. This can be easily seen visually if we consider the change in the brilliance of coals in the process of changing them. No special instruments are required to notice that the brilliance of anthracite, for example, is much higher than that of brown coal. Reflectivity is closely related to the internal structure of a substance, namely, the degree of packing of particles in a substance. That's what she depends on. Of course, the study of the degree of catagenesis by reflectivity is carried out using special equipment For example, the POOS-I setup consists of a polarizing microscope, an optical attachment, a photomultiplier tube (PMT) and a recording device. When conducting a study, photocurrents caused by light reflected from the surface of the sample and the standard are compared.
So, vitrinite, or rather its reflectivity, was taken as the standard for research. It is measured using various photometers and standards in air and immersion medium with strictly perpendicular light incidence on a well-polished sample surface. Measurements are carried out only in a narrow wavelength range: from 525 to 552 nm. This limitation is related to technical specifications device. A wavelength of 546.1 nm is taken as the standard, but small fluctuations around this value have practically no noticeable effect on the measurement value. The sample is fixed on the microscope stage and stopped so that its surface is perpendicular to the axis of the optical attachment. As mentioned above, we measure the intensity of the reflected light alternately at the sample and the standard using a PMT. By definition, reflectivity is the ability to reflect some of the light that hits a surface. If we translate this into numerical language, then this is the ratio of reflected light to incident.
Which can be written as:
Where I1 is the reflected light intensity and I2 is the incident light intensity. In practice, when carrying out measurements, the formula is used
Here R is the desired reflection index, d is the reading of the device when measuring the test substance, and R1, respectively, is the reflectance of the standard and d1 is the reading of the device when measuring the standard. If you set the receiver device to zero for the reference, then the formula simplifies to R=d.
In addition to vitrinite, other OM microcomponents are also used for measurements. Some of them have the property of reflectivity anisotropy. Three measurement parameters are usually used: Rmax Rmin Rcp. The increase in vitrinite anisotropy during catagenesis is mainly due to the process of gradual ordering of aromatic humic micelles associated with an increase in pressure with increasing immersion depth. Measurements in the case of an anisotropic preparation are conceptually no different from the measurement of a homogeneous sample, but several measurements are carried out. The microscope stage rotates 360? at intervals of 90?. Two positions with the maximum reflectivity and two with the minimum are always detected. The angle between each of them is 180?. Measurements are made for several rock fragments and the average value is calculated later. As the arithmetic mean of the averages of the maximum and minimum measurements:
You can immediately determine the average value by choosing a rotation angle of 45? from the maximum or minimum value, but this measurement is valid only when studying a weakly transformed OF.
When conducting research, there are several problems associated with the technology. For example, if we have a rock with a low total content of organic matter, then there is a need for special processing of the sample and its conversion into the form of concentrated polished sections-briquettes. But in the process of obtaining concentrates, the original organic matter is subjected to chemical treatment, which cannot but affect the optical properties of the substance. In addition, information about the structure of the organic matter of the rock is lost. Distortions in the measurements can also be introduced by the fact that the technology of the drug preparation process is not standardized and the readiness of the sample is usually determined visually. The problem is the same physical properties rocks, such as strong mineralization or brittleness of coal, in this case it is necessary to study the reflectivity on the surface area that can be obtained. If the area is chosen correctly, then the surrounding defects practically do not affect the measurements. But fundamentally, the quantitative values of errors practically do not affect the determination of the stage of catagenesis.
Samples are studied, usually under normal air conditions, it is easy, fast. But if you need a detailed study under high magnification, immersion media are used, usually cedar oil. Both measurements are correct and each of them is used, but each in its own specific case. The advantages of measurements in an immersion medium are that they allow one to study particles with a small dimension; in addition, sharpness increases, which makes it possible to diagnose the degree of catagenesis in more detail.
An additional difficulty in research is the diagnosis of OM microcomponents, since they are usually determined in transmitted light. While the reflectivity is obviously in the reflected. That's why. Usually, two methods are combined in the research process. That is, transmitted and reflected light are alternately used to study the same DOM fragment. For this, polished sections are usually used on both sides. In them, after viewing and determining the microcomponent in transmitted light, the illumination is switched and measurements are taken in reflected light.
Vitrinite can be used not only to determine the degree of transformation of organic matter, but also to determine its relationship to the rock. In syngenetic vitrinite, the fragments are usually elongated, the particles are parallel to the bedding planes, and usually have a cellular structure. If we are dealing with vitrinite particles of a rounded, rounded shape, then most likely this is a redeposited substance.
The measurement of vitrinite reflectance Ro% is one of the most common methods for assessing the degree of OM maturation in sediments. The reflectivity of vitrinite is measured as the ratio of the intensities of the reflected and incident light beams. According to the physical laws of reflection and refraction of light,
The fraction of intensity, Ro, of a beam of monochromatic light that is normally reflected from flat surface a piece of vitrinite with a refractive index n, immersed in oil with a refractive index, n o (or in air with an index of n a), is equal to:
The refractive indices n and n o are determined by the integral temperature history of the vitrinite sample, i.e. function T(t). The method is based on the idea that during coalification, vitrinite changes its reflectivity from Ro = 0.25% at the peat stage to Ro = 4.0% at the anthracite stage (Lopatin, Emets, 1987). The huge factual material accumulated to date makes it possible to identify certain stages of maturation by the measured values of Ro%. In this case, variations in the values of Ro% for different types of OM are possible, as well as depending on the content of impurities in the OM. Thus, Ro = 0.50% approximately corresponds to the beginning of the main stage of oil formation for high-sulfur kerogens, while Ro = 0.55 - 0.60% - the same stage for type I and II kerogens (see below), and Ro = 0.65 - 0.70% - for type III kerogens (Gibbons et al., 1983; Waples 1985). One of the variants of the supposed correspondence of the Ro% values to the main stages of OM maturation and the calculated values of the temperature-time index (TTI), discussed below, can be seen in tables 1-7a, as well as on rice. 1-7. Correspondence of stages of catagenesis to Ro values given in the table is based on the correlation between the calculated Temperature-Time Indices (TTI) and Ro% values measured in different basins of the world, and is approximate. However, it is widely used in the literature and is discussed in more detail in section 7-5-1. For the convenience of orientation in various scales of OM catagenesis, Tables 1-7b also provide a scale for the correspondence of values
Table 1-7a. Correspondence of Ro% and TWI values to the stages of OS catagenesis(Waples, 1985)
reflectivity of vitrinite %Ro to the maturity stages of organic matter, accepted in Russian petroleum geology.
Table 1-7b. Correspondence of Ro% values to the stages of OM catagenesis accepted in Russian petroleum geology(Parparova et al., 1981)
Diagenesis: DG3, DG2 and DG1 ------ Ro< 0.25%
Protocatagenesis: PC1 (0.25 £ Ro £ 0.30%)
PC2 ((0.30 £ Ro £ 0.42%)
PC2 ((0.42 £ Ro £ 0.53%)
Mesocatagenesis: MK1 (0.53 £ Ro £ 0.65%)
MK2 ((0.65 £ Ro £ 0.85%)
MK3 ((0.85 £ Ro £ 1.15%)
MK4 ((1.15 £ Ro £ 1.55%)
MK5 ((1.55 £ Ro £ 2.05%)
Apocatagenesis: AK1 (2.05 £ Ro £ 2.50%)
AK2 ((2.50 £Ro £3.50%)
AK3 ((3.50 £ Ro £ 5.00%)
AK4 ((Ro > 5.00%)
Let us briefly talk about some problems associated with the use of %Ro measurements to assess the degree of OM catagenesis. They are associated primarily with the difficulty of separating vitrinite macerals from OM of sedimentary rocks due to their great diversity. Using the reflectivity of vitrinite to control paleotemperature conditions is possible, generally speaking, only on the basis of vitrinite from coal seams and, with less reliability, vitrinite from continental (“terrestrial”) parent OM in clays with an organic carbon content not exceeding 0.5%. But even in these continental (terrestrial) series, care should be taken, since in rocks such as sandstones, the main part of OM can be processed and changed (Durand et al. 1986). It is also necessary to take into account the fact that, in any case, for Ro > 2%, the reflectivity will also depend on pressure. Care should also be taken in extending the concept of vitrinite to marine and lacustrine rock series, since in such rocks the particles whose reflectance is measured are rarely vitrinites of higher plants and in most cases
Rice. 1-7. Correlation of vitrinite reflectivity, Ro%, and degree of coalification with other maturity indices and with the position of oil and gas generation and decomposition zones Top: after (Kalkreuth and Mc Mechan, 1984), bottom after (Tissot et al., 1987).
are bituminoids from plankton, mistaken for vitrinite (Waples, 1985; Durand et al. 1986). According to thermophysical properties, they differ from vitrinite. A similar problem exists for the continental (terrestrial) rocks of the Cambrian-Ordovician and older ages. They cannot contain vitrinite, since higher plants did not exist then. In all red formations, OM is oxidized. In limestone, vitrinites are less common and, if present, their reflectivity may differ from that of normal vitrinite of the same degree of coalification (Buntebarth and Stegena, 1986).
Certain errors in this method for assessing OM catagenesis will also arise due to significant scatter in the measured Ro values, as well as due to the fact that in the basin section there will always be horizons in which vitrinite isolation is difficult or impossible at all. For example, at low maturity levels, the isolation of vitrinite macerals is a big problem, and therefore the reliability of Ro measurements for values less than 0.3 - 0.4% is extremely low (Waples et al. 1992). The dependence of the reflectivity of vitrinite on the initial chemical composition of vitrinite will be significant (Durand et al. 1986). This explains the fact that a large spread in Ro% values is often observed even within the same basin (Tissot et al. 1987). In order to make a minimum error due to variations in the chemical composition of vitrinite, Ro% measurements are carried out on samples of regular vitrinite isolated by standard procedure from organic matter of continental origin. It is not recommended to use equivalent types of vitrinite in OM types I and II when creating universal scales for the correspondence of Ro% values to the degrees of OM conversion (Tissot et al. 1987).
And yet, with a reasonable consideration of the comments made, the method for assessing the maturity level of OM and controlling through it the paleotemperature conditions of sedimentary strata subsidence by measuring the reflectivity of vitrinite is currently one of the most reliable and common methods in the practice of analyzing oil and gas basins.
7.3 Using %Ro measurements and other methods to estimate maximum rock temperatures in the history of basin subsidence
Initially, vitrinite reflectance measurements were used to estimate the maximum temperatures Tmax in the history of subsidence of suites. For such purposes, a number of methods have been used and are being used in geological studies, such as (Yalcin et al., 1997): 1) estimates of T max by the level of maturity of OM (degree of coalification, vitrinite reflectivity; 2) estimates based on mineralogical changes during diagenesis of clay minerals and crystallization of illite; 3) methods based on the analysis of liquid inclusions, for example, liquid homogenization temperature; 4) geothermometers based on specific chemical reactions, for example, characterizing the equilibrium of stable isotopes (Hoefs, 1987) or the equilibrium states of the SiO 2 -Na-K-Ca system (Ellis and Mahon, 1977); 5) Fizzion-track analysis (analysis of the distribution of traces from the fission of radioactive elements in appatite; Green et al., 1989; 1995); 6) based on a combination of determinations of the radiometric age of such radiometric systems as K-Ar, Rb-Sr and U, which are closed at various temperatures(Buntebarch and Stegena, 1986). Since paleotemperature estimates are still widely used in the geological literature, we will briefly characterize each of these methods. Let's start the presentation with estimates of the maximum temperatures of rocks from the values of the reflectivity of vitrinite.
It should be noted right away that the development of methods for estimating maximum temperatures in the history of subsidence of sedimentary suites (Tmax) is due to the fact that in the 70s and 80s of the last century, many researchers considered temperature as the main and, in fact, the only factor in the evolution of sedimentary OM maturity. The influence of time on the process of OM maturation was neglected in this case. It was believed that the measured (or calculated) values of vitrinite reflectance %Rо should reflect the maximum temperatures of the rocks in the history of their subsidence. Following such views, various correlations were proposed between the values of T max and the reflectivity of rock vitrinite in air % R a and in oil % Ro . For example, in the works of Ammosov et al. (1980) and Kurchikov (1992) it is proposed to estimate the values of T max from the measured values of %R a from the ratio
10×R a (%) = 67.2× (7-1)
For samples of carbonaceous interlayers in rocks, from the ratio
10×R a (%) = 67.2× (7-2)
For sandstones and siltstones and according to the equation
10×R a (%) = 67.2× (7-3)
For clays and mudstones. In the above expressions, T max is expressed in °C. Price (Price, 1983) also believed that a time of one and even more million years does not have a noticeable effect on the process of OM maturation and, based on this, proposed a relationship similar to (7-1) - (7-3), relating T max with reflectivity of vitrinite in oil (%Ro):
T max (°С) = 302.97×log 10 Ro(%) + 187.33 (7-4)
Several similar relationships have been considered by K. Barker (Barker and Pawlevicz, 1986; Barker, 1988, 1993). The first of these (Barker and Pawlevicz, 1986):
ln Ro(%) = 0.0078×T max (°С) - 1.2 (5)
was based on 600 measurements of T max in 35 wells in various basins of the world. According to the authors, it is valid in the temperature range 25 £ T max £ 325°C and vitrinite reflectivity 0.2% £ Ro £ 4.0%. K. Barker (Barker, 1988) proposed a relationship that describes situations with a constant rate of heating of rocks when immersed in a basin:
T max (°С) = 104×ln Ro(%) + 148. (7-6),
and based on a kinetic model of vitrinite maturation (Burnham and Sweeney, 1989). M. Johnson et al. (Johnsson et al., 1993), analyzing this formula, notice that it describes the situation with heating rates V = 0.1 – 1 °C/mcm rather well. years, but for rates V = 10 – 100 °C/m.y. years underestimates the values of T max in the region of Ro< 0.5% и переоценивает их при Ro >2%. In his later work, Barker (Barker, 1993) proposed another version of the correlation between T max and % Ro, which does not contain restrictions on the rate of rock heating:
T max (°C) = [ln(Ro(%) / 0.356)] / 0.00753 (7-7)
Thus, quite a lot of correlation ratios T max - %Ro are proposed in the literature. On rice. 2-7 they are compared with each other according to the results of estimates of T max for values of 0.4% £ Ro £ 4.0%.
Rice. 2-7. Relationships relating the maximum temperature Tmax in the history of rock subsidence with the measured values of the reflectivity of vitrinite in oil %Ro, according to various literature sources: 1 (for coals), 2 (for sandstones and siltstones), 3 (for clays and mudstones) - ( Ammosov et al., 1980; Kurchikov, 1992); 4 - (Price, 1983); 5 - (Barker and Pawlevicz, 1986); 6 - (Barker and Pawlevicz, 1986); 7 - (Barker, 1993); 8 - according to the temperature of homogenization of liquid inclusions (Tobin and Claxton, 2000).
From this figure, a significant scatter in the values of T max corresponding to fixed values of Ro is obvious, which reaches 60 - 100°C for a maturity of Ro ³ 0.7%. This scatter unambiguously indicates that the temperature value (even if the maximum one) alone cannot determine the maturity of OM in rocks, and that the temperature holding time plays a significant role in the maturation of OM. It is possible that in certain Ro intervals and under special sedimentation conditions (such as those that provide a constant rate of rock heating), some of the above ratios describe the situation quite well, but as studies show (see below), the same values of %Ro can be achieved, for example, at lower temperatures but with longer rock holding times (see below). For this reason, there is always a basin and formation with a corresponding interval of maturity and temperatures, for which estimates according to relations (7-1) - (7-7) will lead to noticeable errors. This circumstance had the consequence that the popularity of the written ratios has noticeably decreased over the past 10-15 years.
Another common method for assessing the paleotemperatures of rocks in basins is the determination of Tmax by analyzing the composition of fluids trapped in the rock matrix during diagenesis. The application of the method is possible under the following conditions (Burruss 1989): 1) the inclusion is a single-phase liquid, 2) the volume of this liquid does not change after it is captured by the rock, 3) its composition also remained unchanged, 4) the effect of pressure on the composition of the liquid is known in advance, 5 ) the time and mechanism of liquid trapping are also known. These conditions suggest that a certain amount of caution is required in the application of the method (Burruss 1989). First, detailed petrographic studies are needed to establish the relative time of formation of the liquid inclusion. Secondly, a thorough analysis of the tectonic development of the area and the history of subsidence of the basin is needed to detail the history of the host rocks. It is also necessary to analyze the phase behavior and chemical composition of the trapped liquid. But even after this, two important problems remain - one related to the assumption that the chemical composition of the liquid remains unchanged after its capture by the rock matrix (there is convincing evidence that this is not always the case), and the other related to determining the magnitude and type of pressure that existed during the period fluid containment - whether it was lithostatic or hydrostatic (Burruss 1989). If all these problems are solved, the temperature of the rock at the time of liquid capture is determined by the corresponding P-T equilibrium diagram of the liquid and solid phases of the test substance. In the development of this method, Tobin and Claxton (Tobin and Claxton, 2000) proposed to use the correlation between the homogenization temperature of liquid inclusions T hom and the reflectivity of vitrinite Ro% (Fig. 2-7):
Ro% = 1.9532 ´ log T hom – 2.9428 (7-8)
They found that when using an “ideal” series of measurements, relation (7-8) is satisfied with a correlation coefficient of 0.973 and a data variance of less than 0.12% Ro. If the entire series of world data is used, then the relation of the form:
Ro = 2.1113 ´ log T hom – 3.2640 (7-9)
will be performed with a correlation coefficient of 0.81 and a maximum data variance of less than 0.32% Ro (Tobin and Claxton, 2000). The homogenization temperature T hom is often used as an estimate of the maximum rock temperature T max during its subsidence in the basin. However, fig. 2-7 shows that the curve constructed according to the formula (7-9) differs markedly from the estimates of T max according to the formulas (7-1) - (7-7), crossing the rest of the lines in Fig. 2-7. It clearly underestimates the temperatures for Ro< 1.5% и даёт нереально высокие значения при Ro >2% (Th = 540, 930, and 1600°C for Ro=2.5, 3, and 3.5%, respectively).
Figure 3-7 Change in d 13 C isotope ratio with depth for the Anadarko Basin gas field (USA; Price, 1995).
In a number of works (Rooney et al., 1995; Price, 1995, etc.), it is proposed to use the change in the carbon isotope composition during OM catagenesis to estimate the temperature of hydrocarbon generation. (Figure 3-7). Results of experiments on the generation of gases of OM type II (source rocks of the Delaware and Val Verde basins in West Texas) at a constant rock heating rate of 1°C/min (left rice. 4-7; Rooney et al., 1995) show a noticeable change in the isotopic composition of gases
Rice. 4-7. Gas generation temperature and isotopic ratio d 13 C for methane (d 13 C 1), ethane (d 13 C 2) and propane (d 13 C 3) generated from type II kerogen source rocks of the Delaware and Val Verde basins in west Texas at rock heating rate 1°C/min (left figure, after Rooney et al., 1995) and Isotope ratio d 13 C for methane generated at different temperatures during hydroid pyrolysis of rock samples with OM various types(right figure, after Price, 1995).
with temperature and thus confirm the fundamental possibility of using this dependence to estimate the temperature of generation of gases of OM of this type. The same is evidenced by the results of hydroid pyrolysis of rock samples with various types of organic matter, shown in the left figure. 4-7. They also clearly demonstrate the change in the d 13 C isotope ratio for methane generated at different temperatures (Price, 1995). However, these experiments also indicate an extremely high sensitivity of changes in d 13 C to variations in the composition and type of OM, which is why the method can be applied only after a detailed analysis of the OM composition and obtaining the corresponding dependences for the analyzed type of substance. The wide variation in d 13 C values with depth shown in fig. 3-7 for a typical section of a sedimentary basin is mainly caused by variations in the composition and type of OM in the rocks of the macro and micro layers of the section. Such a scatter severely limits the reliability of temperature estimates based on isotope ratios in gases from real sedimentary sections.
The process of converting smectite to illite in clay minerals is also sometimes used to control paleotemperature conditions in basins. However, rice. 5-7 shows that the temperature ranges characteristic of the process are quite wide. This variation in temperature is not surprising, since laboratory research show that the process of converting smectite to illite is driven by a 6th order kinetic reaction (Pytte and Reynolds, 1989) and hence time influences the rates of these transitions along with temperature. These reactions will be considered in more detail in the final section of this chapter, but here we note that reasonable estimates of the temperature of the transition of smectite to illite are possible only for the isothermal version of the transformation of minerals, but even then the error of the method will be noticeable.
Fig. 5-7 Transformation of clay minerals according to the analysis of samples from 10 wells in the North Sea (Dypvik, 1983). The processes of disappearance of smectite and illite layers of different levels in mixed-layer smectite-illite clay minerals are related to the temperatures and reflectivity of vitrinite.
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FEDERAL AGENCY FOR TECHNICAL REGULATION AND METROLOGY
NATIONAL
STANDARD
RUSSIAN
FEDERATION
MEDICAL PRODUCTS FOR DIAGNOSIS
IN VITRO
Information provided by the manufacturer with in vitro diagnostic reagents used for staining in biology
In vitro diagnostic medical devices - Information supplied by the manufacturer with in vitro diagnostic reagents for staining in biology (IDT)
Official edition
Standartinform
Foreword
Goals and principles of standardization in Russian Federation installed federal law dated December 27, 2002 No. 184-FZ “On technical regulation”, and the rules for the application of national standards of the Russian Federation - GOST R 1.0-2004 “Standardization in the Russian Federation. Basic Provisions»
About the standard
1 PREPARED BY the Laboratory of Problems of Clinical and Laboratory Diagnostics of the Research Institute of Public Health and Health educational institution higher vocational education First Moscow State Medical University. I. M. Sechenov” of the Ministry of Health of the Russian Federation on the basis of its own authentic translation into Russian of the international standard specified in paragraph 4
2 INTRODUCED by the Technical Committee for Standardization TK 380 "Clinical laboratory research and medical devices for in vitro diagnostics"
3 APPROVED AND INTRODUCED BY Order federal agency on technical regulation and metrology dated October 25, 2013 No. 1201-st.
4 This standard is identical to the international standard ISO 19001:2002 “Medical devices for in vitro diagnostics. Information supplied by the manufacturer with in vitro diagnostic reagents for staining in biology” (ISO 19001:2002 “/l vitro diagnostic medical devices - Information supplied by the manufacturer with in vitro diagnostic reagents for staining in biology”).
The name of this standard has been changed relative to the name of the specified international standard to bring it into line with GOST R 1.5 (subsection 3.5).
5 INTRODUCED FOR THE FIRST TIME
The rules for the application of this standard are established in GOST R 1.0-2012 (section 8). Information about changes to this standard is published in the annually published information index "National Standards", and the text of changes and amendments - in the monthly published information indexes "National Standards". In case of revision (replacement) or cancellation of this standard, a corresponding notice will be published in the monthly published information index "National Standards". Relevant information, notification and texts are also placed in information system general use - on the official website of the Federal Agency for Technical Regulation and Metrology on the Internet (gost.ru)
© Standartinform, 2014
This standard cannot be fully or partially reproduced, replicated and distributed as an official publication without the permission of the Federal Agency for Technical Regulation and Metrology
A.4.2.3.3 Staining procedure
A.4.2.3.3.1 Dewax and rehydrate tissue sections; perform an antigen change (see above staining method)
A.4.2.3.3.2 Incubate with 3% hydrogen peroxide in distilled water for 5
A.4.2.3.3.3 Wash with distilled water and place in TBS for 5 min.
A.4.2.3.3.4 Incubate with monoclonal mouse anti-human estrogen receptor optimally diluted in TBS (see A.4.2.3) for 20 min to 30 min.
A.4.2.3.3.5 Wash with TBS and place in the TBS bath for 5 min.
A.4.2.3.3.6 Incubate with biotinylated goat anti-mouse/rabbit immunoglobulin working solution for 20 min to 30 min.
A.4.2.3.3.7 Wash with TBS and place in the TBS bath for 5 min.
A.4.2.3.3.8 Incubate with the working solution of the Streptavidin-biotin/horseradish peroxidase complex for 20 to 30 minutes.
A.4.2.3.3.9 Wash with TBS and place in the TBS bath for 5 min.
A.4.2.3.3.10 Incubate with DAB solution for 5-15 min (use gloves when handling DAB).
A.4.2.3.3.11 Rinse with distilled water.
A.4.2.3.3.12 Counterstain with hematoxylin solution for 30 s.
A.4.2.3.3.13 Rinse with tap water for 5 min.
A.4.2.3.3.14 Rinse with distilled water for 5 min.
A.4.2.3.3.15 Dehydrate with 50% v/v ethanol for 3 min, then 3 min with 70% v/v and finally 3 min with 99% v/v.
A.4.2.3.3.16 Wash in two changes of xylene, 5 minutes each. A.4.2.3.3.17 Work up into a synthetic hydrophobic resin.
A.4.2.3.4 Suggested dilutions
Optimal staining can be obtained by diluting the antibody in TBS pH 7.6 mixed by volume from (1 + 50) to (1 + 75) µl when examined on formalin-fixed paraffin-embedded human breast cancer sections. The antibody can be diluted with TBS, mixed in volumes from (1 + 50) to (1 + 100) µl, for use in APAAP technology and avidin-biotin methods, in the study of acetone-fixed sections of frozen breast cancer tissue.
A.4.2.3.5 Expected results
The antibody intensely labels the nuclei of cells known to contain big number estrogen receptors, for example, epithelial and myometrial cells of the uterus and normal and hyperplastic epithelial cells of the mammary glands. Staining is predominantly localized in the nuclei without staining of the cytoplasm. However, cryostat sections containing small or undetectable amounts of estrogen receptors (eg, intestinal epithelium, heart muscle cells, brain and connective tissue cells) show negative results with antibody. The antibody targets breast carcinoma epithelial cells that express the estrogen receptor.
Fabric dyeing depends on the handling and processing of the fabric prior to dyeing. Improper fixation, freezing, thawing, rinsing, drying, heating, cutting, or contamination with other tissues or fluids may cause artifacts or false negative results.
A.5 Demonstration of 7-cells by flow cytometry
CAUTION - The reagent contains sodium azide (15 mmol/L). NaN 3 can react with lead or copper to form explosive metal azides. When removed, rinse with plenty of water.
A.5.1 Monoclonal mouse anti-human G-cells
The following information applies to monoclonal mouse anti-human 7-kpets:
a) product identity: monoclonal mouse anti-human 7-cells, CD3;
b) clone: UCHT;
c) immunogen: human childhood thymocytes and lymphocytes from a patient with Sezary's disease;
d) source of antibodies: purified monoclonal mouse antibodies;
e) specificity: the antibody reacts with T cells in the thymus, bone marrow, peripheral lymphoid tissue and blood. Most tumor T cells also express the CD3 antigen, but it is absent in non-T cell lymphoid tumors. Consistent with the model of antigen synthesis in normal thymocytes, the earliest site of detection in tumor cells is the cytoplasm of the cell;
f) Composition:
0.05 mol/l Tris/HCI buffer, 15 mmol/l NaN 3 , pH = 7.2, bovine serum albumin, mass fraction 1
lg isotype: IgGI;
Ig purification: protein A Sepharose column;
Purity: mass fraction approximately 95%;
Conjugate molecule: fluorescein isothiocyanate isomer 1 (FITC);
- (NR)-ratio: £ 495 nm / £ 278 nm = 1.0 ± 0.1 corresponding to a molar ratio of FITC / protein of approximately 5;
e) handling and storage: stable for three years after isolation at temperatures from 2 °C to 8
A.5.2 Intended use
A.5.2.1 General
The antibody is intended for use in flow cytometry. The antibody can be used for the qualitative and quantitative detection of T cells.
A.5.2.2 Type(s) of material
The antibody can be applied to fresh and fixed cell suspensions, acetone-fixed cryostat sections, and cell smears.
A.5.2.3 Procedure for testing antibody reactivity for flow cytometry
The details of the methodology used by the manufacturer are as follows:
a) Collect venous blood in a tube containing an anticoagulant.
b) Isolate mononuclear cells by centrifugation on a separation medium; otherwise, lyse the erythrocytes after the incubation step in d).
c) Wash mononuclear cells twice with RPMI 1640 or phosphate buffered saline (PBS) (0.1 mol/l phosphate, 0.15 mol/l NaCl, pH = 7.4).
d) To 10 µl of FITC-conjugated monoclonal mouse anti-human T cells, CD3 reagent, add a cell suspension containing 1-10 e cells (usually about 100 ml) and mix. Incubate in the dark at 4°C for 30 min [R-Phycoerythrin-conjugated (RPE) antibody should be added at the same time for double staining].
f) Wash twice with PBS + 2% bovine serum albumin; resuspend the cells in the appropriate fluid for flow cytometer analysis.
f) Another monoclonal antibody conjugated with FITC (fluorescein isothiocyanate) is used as a negative control.
e) Fix the precipitated cells by mixing with 0.3 ml of paraformaldehyde, 1% mass fraction in PBS. When stored in the dark at 4°C, fixed cells can be maintained for up to two weeks.
h) Analyze on a flow cytometer.
A.5.2.4 Suggested dilution
The antibody should be used for flow cytometry in concentrated form (10 µl/gest). For use on cryostat sections and cell smears, the antibody must be mixed with a suitable diluent in a volume ratio of (1 + 50) µl.
A.5.2.5 Expected results
The antibody detects the CD3 molecule on the surface of the T cells. When evaluating the staining of cryostat sections and cell smears, the reaction product should be localized on the plasma membrane.
Fabric dyeing depends on the handling and processing of the fabric prior to dyeing. Improper fixation, freezing, thawing, rinsing, drying, heating, sectioning, or contamination with other tissues or fluids may cause artifacts or false negative results.
Appendix YES (reference)
Information on the compliance of reference international and European regional standards with the national standards of the Russian Federation
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Table YES.1 |
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NATIONAL STANDARD OF THE RUSSIAN FEDERATION
MEDICAL DEVICES FOR IN VITRO DIAGNOSTICS Information provided by the manufacturer with in vitro diagnostic reagents used for staining in biology
In vitro diagnostic medical devices. Information supplied by the manufacturer with in vitro diagnostic reagents for staining in biology
Introduction date - 2014-08-01
1 area of use
This International Standard specifies requirements for information supplied by manufacturers with reagents used for staining in biology. The requirements apply to manufacturers, suppliers and sellers of dyes, dyes, chromogenic reagents and other reagents used for staining in biology. The requirements for information supplied by manufacturers, as set out in this International Standard, are necessary condition obtaining comparable and reproducible results in all areas of staining in biology.
This standard uses normative references to the following international and European regional standards:
ISO 31-8, Quantities and units. Part 8. Physical chemistry and molecular physics (ISO 31-8, Quantities and units - Part 8: Physical chemistry and molecular physics)
EH 375:2001, Information supplied by the manufacturer with in vitro diagnostic reagents for professional use
EH 376:2001, Information supplied by the manufacturer with in vitro diagnostic reagents for self-testing
Note - When using this standard, it is advisable to check the validity of reference standards in the public information system - on the official website of the Federal Agency for Technical Regulation and Metrology on the Internet or according to the annual information index "National Standards", which was published as of January 1 of the current year, and on issues of the monthly information index "National Standards" for the current year. If an undated referenced reference standard has been replaced, it is recommended that the current version of that standard be used, taking into account any changes made to that version. If the reference standard to which the dated reference is given is replaced, then it is recommended to use the version of this standard with the year of approval (acceptance) indicated above. If, after the approval of this standard, a change is made to the referenced standard to which a dated reference is given, affecting the provision to which the reference is given, then this provision is recommended to be applied without taking into account this change. If the reference standard is canceled without replacement, then the provision in which the reference to it is given is recommended to be applied in the part that does not affect this reference.
3 Terms and definitions
In this standard, the following terms are used with their respective definitions:
3.1 information supplied by the manufacturer all printed, written, graphic or other information supplied with or accompanying the IVD reagent
3.2 label any printed, written or graphic information that appears on a package
Official edition
3.3 in vitro diagnostic reagent reagent used alone or in combination with others medical devices for in vitro diagnostics intended by the manufacturer for in vitro studies of substances of human, animal or plant origin for the purpose of obtaining information related to the detection, diagnosis, monitoring or treatment of physiological conditions, health conditions or diseases or congenital anomalies.
3.4 staining imparting color to a material by reaction with a dye or chromogenic reagent
3.5 dye (dye) colored organic compound which, when dissolved in a suitable solvent, is capable of imparting color to a material
NOTE The physical nature of color is selective absorption (and/or emission) in the visible region of the electromagnetic spectrum between 400 and 800 nm. Dyes are molecules with large systems of delocalized electrons (bound tt-electron systems). The light absorption characteristics of colorants are represented by an absorption spectrum in the form of a diagram in which light absorption and wavelength are compared. The spectrum and wavelength at maximum absorption depend on the chemical structure of the dye, the solvent and on the conditions of the spectral measurement.
3.6 stain
NOTE The paint may be prepared by direct dissolution of the coloring matter in a solvent or dilution of the prepared stock solution with suitable agents.
3.6.1 stock solution of stain
NOTE Stability means that the properties of a colorant remain constant even in the presence of other colorants.
3.7 chromogenic reagent reagent that reacts with chemical groups present or elicited in cells and tissues to form a colored compound in situ
EXAMPLE Typical chromogenic reagents:
a) diazonium salt;
b) Schiff's reagent.
3.8 fluorochrome reagent that emits visible light when irradiated with excitation light of a shorter wavelength
3.9 antibody specific immunoglobulin produced by B-lymphocytes in response to exposure to an immunogenic substance and capable of binding to it
Note - An immunogenic substance molecule contains one or more parts with a characteristic chemical composition, epitope.
3.9.1 polyclonal antibody mixture of antibodies capable of reacting specifically with a particular immunogenic substance
3.9.2 monoclonal antibody antibody capable of specifically reacting with a single epitope of a specified immunogenic substance
3.10 nucleic acid probe
3.11 lectin protein of non-immunogenic origin with two or more binding sites that recognizes and binds to specific saccharide residues
4 Requirements for information supplied by the manufacturer
4.1 General requirements
4.1.1 Information provided by the manufacturer with reagents used for staining in biology
Information supplied by the manufacturer with reagents used for staining in biology shall be in accordance with ISO 31-8, ISO 1000, EN 375 and EN 376. Special attention attention should be paid to the warnings given in EN 375. In addition, if applicable, the requirements specified in 4.1.2, 4.1.3 and 4.1.4 should be applied to the various reagents used for staining in biology.
4.1.2 Product name
The product name must include the CAS registration number and the dye name and index number, if applicable.
Note 1 The registry numbers in the CAS are the registry numbers in the Chemical Reference Service (CAS). They are the numerical code numbers of substances that have received an index in the Chemical Reference Service assigned to chemicals.
Note 2 - The paint index gives a 5-digit number, C.I number. and a specially composed name for most dyes.
4.1.3 Reagent description
The description of the reagent should include the relevant physicochemical data, followed by the details specific to each lot. The data must contain at least the following information:
a) molecular formula including counterion;
b) molar mass (g/mol) explicitly stated, with or without the inclusion of a counter-ion;
c) limits for interfering substances;
For painted organic compounds data must contain:
d) molar absorbance (instead, the content of the pure colorant molecule may be given, but not the content of the total colorant);
e) wavelength or number of waves at maximum absorption;
f) data from thin layer chromatography, high performance liquid chromatography or high performance thin layer chromatography.
4.1.4 Intended use
A description should be provided providing guidance on staining in biology and quantitative and qualitative procedures (if applicable). The information must include information regarding the following:
a) type(s) of biological material, handling and pre-staining processing, e.g.:
1) whether cell or tissue samples can be used;
2) whether frozen or chemically fixed material can be used;
3) protocol for tissue handling;
4) what fixing medium can be applied;
b) details of the appropriate reaction procedure used by the manufacturer to test the reactivity of a dye, dye, chromogenic reagent, fluorochrome, antibody, nucleic acid probe or lectin used for staining in biology;
c) the result(s) expected from the reaction procedure on the intended type(s) of material in the manner intended by the manufacturer;
d) comments on the appropriate positive or negative tissue control and on the interpretation of the result(s);
4.2 Additional requirements to reagents of specific types
4.2.1 Fluorochromes
Regardless of the type of application, fluorochromes proposed for staining in biology must be accompanied by the following information:
a) selectivity, such as a description of the target(s) that can be demonstrated using specific conditions; wavelengths of excitation and emission light; for antibody-bound fluorochromes, the fluorochrome/protein ratio (F/B).
4.2.2 Metal salts
Where metal-containing compounds are proposed for use in a metal-absorbing technique for staining in biology, the following additional information must be provided:
systematic name; purity (no impurities).
4.2.3 Antibodies
Antibodies proposed for staining in biology must be accompanied by the following information:
a) a description of the antigen (immunogenic substance) against which the antibody is directed and, if the antigen is determined by the cluster of the differentiation system, the CD number. The description should contain, if applicable, the type of macromolecule to be detected, part of which is to be detected, the cellular localization and the cells or tissues in which it is found, and any cross-reactivity with other epitopes;
b) for monoclonal antibodies, clone, method of formation (tissue culture supernatant or ascitic fluid), immunoglobulin subclass, and light chain identity;
c) for polyclonal antibodies, the host animal and whether whole serum or an immunoglobulin fraction is used;
a description of the form (solution or lyophilized powder), the amount of total protein and specific antibody, and for a solution, the nature and concentration of the solvent or medium;
e) if applicable, a description of any molecular binders or excipients added to the antibody;
a statement of purity, purification technique, and methods for detecting impurities (eg, Western blotting, immunohistochemistry);
4.2.4 Nucleic acid probes
Nucleic acid probes proposed for staining in biology must be accompanied by the following information:
the sequence of bases and is the probe one- or two-stranded; the molar mass of the probe or the number of bases and, if applicable, the number of fractions (in percent) of guanine-cytosine base pairs;
used marker (radioactive isotope or non-radioactive molecule), point of attachment to the probe (3" and/or 5") and percentage of substance in percent of the labeled probe; detectable gene target (DNA or RNA sequence);
e) a description of the form (lyophilized powder or solution) and amount (pg or pmol) or concentration (pg/ml or pmol/ml), if applicable, and, in the case of a solution, the nature and concentration of the solvent or medium;
f) claims of purity, purification procedures and methods for detecting impurities, eg high performance liquid chromatography;
Annex A (informative)
Examples of information provided by the manufacturer with reagents commonly used
in biological staining techniques
A.1 General
The following information is an example of procedures and should not be construed as the only way procedure to be carried out. These procedures can be used by the manufacturer to test the reactivity of colorants and illustrate how a manufacturer can provide information to comply with this International Standard.
A.2 Methyl green-pyronine Y dye A.2.1 Methyl green dye
The information regarding the colorant methyl green is as follows:
a) product identity:
Methyl green (synonyms: double green SF, light green);
CAS registration number: 22383-16-0;
Name and color index number: basic blue 20, 42585;
b) composition:
Molecular formula, including the counterion: C 2 bH3M 3 2 + 2BF4 ";
Molar mass with (or without) counterion: 561.17 g mol "1 (387.56 g
Mass fraction (content) of methyl green cation: 85%, determined by absorption spectrometry;
Permissible limits for interfering substances, given as mass fractions:
1) water: less than 1%;
2) inorganic salts: less than 0.1%;
3) detergents: not present;
4) colored impurities, including violet crystals: not detectable by thin layer chromatography;
5) indifferent compounds: 14% soluble starch;
d) thin layer chromatography: only one main component is present, corresponding to
methyl green;
e) Handling and storage: Stable when stored in a tightly stoppered brown bottle at room temperature (18°C to 28°C).
A.2.2 Colorant ethyl green
The information related to the colorant ethyl green is as follows:
a) product identity:
1) ethyl green (synonym: methyl green);
2) CAS registration number: 7114-03-6;
3) name and number of the paint index: no name in the paint index, 42590;
b) composition:
1) molecular formula including counterion: C27H 3 5N 3 2+ 2 BF4";
2) molar mass with (or without) counterion: 575.19 g mol" 1 (401.58 g mol" 1);
3) mass fraction of ethyl green cation: 85%, determined using absorption spectrometry;
Water: less than 1%;
Detergents: none;
c) maximum absorption wavelength of the dye solution: 633 nm;
d) thin layer chromatography: only one major component is present, matching ethyl green;
A.2.3 Pyronin Y dye
Pyronin Y coloring matter includes the following information:
a) product identity:
1) pyronin Y (synonyms: pyronine Y, pyronin G, pyronine G);
2) CAS registration number: 92-32-0;
3) name and number in the paint index: no name in the paint index, 45005;
b) composition:
1) molecular formula including counterion: Ci7HigN20 + SG;
2) molar mass with (or without) counterion: 302.75 g mol" 1 (267.30 g mol" 1);
3) mass fraction of pyronin Y cation: 80%, determined using absorption spectrometry;
4) permissible limits of interfering substances, given as mass fractions:
Water: less than 1%;
Inorganic salts: less than 0.1%;
Detergents: none;
Colored impurities, including violet crystals: not detectable by thin layer chromatography;
Indifferent compounds: 19% soluble starch;
c) maximum absorption wavelength of the dye solution: 550 nm;
d) thin layer chromatography: only one major component is present, matching pyronin Y;
e) Handling and storage: Stable when stored in a carefully closed brown glass bottle at room temperature between 18 °C and 28 °C.
A.2.4 Intended use of the methyl green-pyronine Y staining method
A.2.4.1 Type(s) of material
Methyl green-pyronine Y stain is used for staining fresh frozen, waxed or plastic tissue sections. various kinds.
A.2.4.2 Handling and processing before staining Possible fixatives include:
Carnoy's liquid [ethanol (99% v/v) + chloroform + acetic acid (99% v/v) mixed in volumes (60 + 30 + 10) ml] or
Formaldehyde (mass fraction 3.6%) buffered with phosphate (pH = 7.0); routine drying, cleaning, impregnating and coating with paraffin, conventional sectioning with a microtome.
A.2.4.3 Working solution
Prepare a solution of ethyl green or methyl green from an amount corresponding to the mass of 0.15 g of pure colorant, calculated as a colored cation (in the examples above 0.176 g in each case) in 90 ml of hot (temperature 50 ° C) distilled water.
Dissolve an amount corresponding to the mass of 0.03 g of pyronin Y, calculated as the colored cation (0.038 g in the example above) in 10 ml of 0.1 mol/l phthalate buffer (pH = 4.0). Mix the last solution with a solution of ethyl green or methyl green.
A.2.4.4 Stability
The working solution is stable for at least one week when stored in a tightly closed brown glass bottle at room temperature between 18°C and 28°C.
A.2.4.5 Staining procedure A.2.4.5.1 Deparaffinize the sections.
A.2.4.5.2 Wet the sections.
A.2.4.5.3 Stain the sections for 5 min at room temperature at about 22 °C in the working
solution.
A.2.4.5.4 Wash the sections in two changes of distilled water, 2 to 3 s each.
A.2.4.5.5 Shake off excess water.
A.2.4.5.6 Activate in three changes of 1-butanol.
A.2.4.5.7 Transfer directly from 1-butanol to a hydrophobic synthetic resin.
A.2.4.6 Expected result(s)
The following results are expected with the material types listed in A.2.4.1:
a) for nuclear chromatin: green (Karnov's fixative) or blue (formaldehyde fixative); a) for nucleoli and cytoplasm rich in ribosomes: red (Karnov's fixative) or lilac-red (formaldehyde fixative);
c) for cartilage matrix and mast cell granules: orange;
d) for muscles, collagen and erythrocytes: not stained.
A.3 Feulgen-Schiff reaction
A.3.1 Colorant pararosaniline
CAUTION -For R 40: possible risk irreversible effects.
For S 36/37: Protective clothing and gloves required.
The following information applies to the dye pararosaniline.
a) product identity:
1) pararosanilin (synonyms: basic ruby, parafuxin, paramagenta, magenta 0);
2) CAS registration number: 569-61-9;
3) name and index number of paints: basic red 9, 42500;
b) composition:
1) molecular formula including counterion: Ci9Hi 8 N 3 + SG;
2) molar mass with (and without) pritivoion: 323.73 g mol "1 (288.28 g mol" 1);
3) mass fraction of pararosaniline cation: 85%, determined by absorption spectrometry;
4) permissible limits of interfering substances, given as mass fractions:
Water: less than 1%;
Inorganic salts: less than 0.1%;
Detergents: not present;
Colored impurities: methylated pararosaniline homologues may be present in trace amounts as determined by thin layer chromatography, but acridine is absent;
Indifferent compounds: 14% soluble starch;
c) maximum absorption wavelength of the dye solution: 542 nm;
d) thin layer chromatography: one main component is present corresponding to
pararosaniline; methylated homologues of pararosaniline in trace amounts;
e) Handling and storage: Stable when stored in a tightly stoppered brown bottle at room temperature between 18 °C and 28 °C.
A.3.2 Intended use of the Feulgen-Schiff reaction
A.3.2.1 Type(s) of material
The Felgen-Schiff reaction is used for waxed or plastic sections of various types of tissues or cytological material (smear, tissue imprint, cell culture, monolayer):
A.3.2.2 Handling and processing before staining
A.3.2.2.1 Possible fixatives
Possible fixatives include:
a) histology: formaldehyde (mass fraction 3.6%) buffered with phosphate (pH = 7.0);
b) cytology:
1) liquid fixing material: ethanol (volume fraction 96%);
2) air dried material:
Formaldehyde (mass fraction 3.6%) buffered with phosphate;
Methanol + formaldehyde (mass fraction 37%) + acetic acid (mass fraction 100%), mixed in volumes (85 + 10 + 5) ml.
The material fixed in Buin's fixative is unsuitable for this reaction.
Details of the procedure used by the manufacturer to test the reactivity of the chromogenic reagent are given in A.3.2.2.2 to A.3.2.4.
A.3.2.2.2 Pararosaniline-Schiff reagent
Dissolve 0.5 g of pararosaniline chloride in 15 ml of 1 mol/l hydrochloric acid. Add 85 ml of an aqueous solution of K 2 S 2 0 5 (mass fraction 0.5%). Wait 24 hours. Shake 100 ml of this solution with 0.3 g charcoal for 2 min and filtered. Store colorless liquid at a temperature not lower than 5 °C. The solution is stable for at least 12 months in a tightly closed container.
A.3.2.2.3 Wash solution
Dissolve 0.5 g of K 2 S 2 O s in 85 ml of distilled water. 15 ml of 1 mol/l hydrochloric acid are added. The solution is ready for immediate use and can be used within 12 hours.
A.3.2.3 Staining procedure
A.3.2.3.1 Dewax the waxed sections in xylene for 5 min, then wash for 2 min, first in 99% v/v ethanol and then in 50% v/v ethanol.
A.3.2.3.2 Wet plastic sections, deparaffinized waxed sections and cytological material in distilled water for 2 min.
A.3.2.3.3 Hydrolyze the material in 5 mol/l hydrochloric acid at 22 °C for 30 to 60 minutes (the exact hydrolysis time depends on the type of material).
A.3.2.3.4 Rinse with distilled water for 2 min.
A.3.2.3.5 Stain with pararosaniline for 1 h.
A.3.2.3.6 Wash in three successive changes of wash solution of 5 min each.
A.3.2.3.7 Wash twice with distilled water, 5 min each time.
A.3.2.3.8 Dehydrate in 50% v/v ethanol, then 70% v/v, and finally 99% ethanol for 3 min each time.
A.3.2.3.9 Wash twice in xylene for 5 minutes each time.
A.3.2.3.10 Take up in a synthetic hydrophobic resin.
A.3.2.4 Expected results
The following results are expected with the types of materials listed in A.3.2.1:
For cell nuclei (DNA): red.
A.4 Immunochemical demonstration of estrogen receptors
CAUTION - Reagent containing sodium azide (15 mmol/L). NaN 3 can react with lead or copper to form explosive metal azides. When removed, rinse with plenty of water.
A.4.1 Monoclonal mouse anti-human estrogen receptor
The following information relates to the monoclonal mouse anti-human estrogen receptor.
a) product identity: monoclonal mouse anti-human estrogen receptor, clone 1D5;
b) clone: 1D5;
c) immunogen: recombinant human estrogen receptor protein;
d) antibody source: mouse monoclonal antibody delivered in liquid form as tissue culture supernatant;
e) specificity: the antibody reacts with the L/-terminal domain (A/B region) of the receptor. On immunoblotting, it reacts with a 67 kDa polypeptide chain obtained by transforming Escherichia coli and transfecting COS cells with estrogen receptor-expressing plasmid vectors. In addition, the antibody reacts with cytosolic extracts of the luteal endometrium and cells of the MCF-7 human breast cancer line;
f) cross-reactivity: the antibody reacts with rat estrogen receptors;
e) composition: tissue culture supernatant (RPMI 1640 medium containing fetal calf serum) dialyzed against 0.05 mmol/l Tris/HCI, pH = 7.2, containing 15 mmol/l NaN3.
Ig concentration: 245 mg/l;
Ig isotype: IgGI;
Light chain identity: kappa;
Total protein concentration: 14.9 g/l;
h) Handling and storage: Stable for up to three years when stored at 2 °C to 8 °C.
A.4.2 Intended use
A.4.2.1 General
The antibody is used for qualitative and semi-quantitative detection of estrogen receptor expression (eg, breast cancer).
A.4.2.2 Type(s) of material
The antibody can be applied to formalin-fixed paraffin sections, acetone-fixed frozen sections, and cell smears. In addition, the antibody can be used to detect antibodies by enzyme-linked immunosorbent assay (ELISA).
A.4.2.3 Staining procedure for immunohistochemistry
A.4.2.3.1 General
For formalin-fixed paraffin-embedded tissue sections, a variety of sensitive staining techniques are used, including the immunoperoxidase technique, APAAP (alkaline phosphatase anti-alkaline phosphatase) technology, and avidin-biotin methods, such as LSAB (Labeled StreptAvidin-Biotin) methods. Antigen modifications, such as heating in 10 mmol/l citrate buffer, pH=6.0, are mandatory. Slides should not dry out during this processing or during the next immunohistochemical staining procedure. The APAAP method has been proposed for staining cell smears.
Details of the procedure used by the manufacturer on paraffin-embedded formalin-fixed tissue sections to test antibody reactivity for immunohistochemistry are given in A.4.2.3.2 to A.4.2.3.4.
A.4.2.3.2 Reagents
A.4.2.3.2.1 Hydrogen peroxide, 3% by mass in distilled water.
A.4.2.3.2.2 Tris buffer saline (TBS), consisting of 0.05 mol/l Tris/HCI and 0.15 mol/l NaCI at pH =
A.4.2.3.2.3 Primary antibody consisting of a monoclonal mouse anti-human estrogen receptor optimally diluted in TBS (see A.4.2.3.4).
A.4.2.3.2.4 Biotinylated goat anti-mouse/rabbit immunoglobulin, working
Prepare this solution at least 30 minutes, but not earlier than 12 hours before use, as follows:
5 ml TBS, pH = 7.6;
50 µl of biotinylated, affinity-isolated goat anti-mouse/rabbit immunoglobulin antibody in 0.01 mol/l phosphate buffer solution, 15 mmol/l NaN3, sufficient to bring the final concentration to 10-20 mg/ml.
A.4.2.3.2.5 StreptAvidin-biotin/horseradish peroxidase complex (StreptABComplex/HRP), working
Prepare this solution as follows:
5 ml TBS, pH = 7.6;
50 µl StreptAvidin (1 mg/l) in 0.01 mol/l phosphate buffer solution, 15 mmol/l NaN 3 ;
50 µl biotinylated horseradish peroxidase (0.25 mg/l) in 0.01 mol/l phosphate buffer solution, 15 mmol/l NaN 3 ;
A.4.2.3.2.6 Diaminenzidine substrate solution (DAB)
Dissolve 6 mg of 3,3"-in 10 ml of 0.05 mol/l TBS, pH = 7.6. Add 0.1 ml of hydrogen peroxide, 3% mass fraction in distilled water. If precipitation occurs, filter.
A.4.2.3.2.7 Hematoxylin
Dissolve 1 g of hematoxylin, 50 g of aluminum potassium sulfate, 0.1 g of sodium iodate and 1.0 g of citric acid in 750 ml of distilled water. Dilute to 1000 ml with distilled water.
The reflectivity of vitrinite is calculated both in air R а and in oil immersion R o . r . By the value of R o . r is estimated class of coal in the industrial - genetic classification (GOST 25543-88).
On fig. 2.1 shows the relationship between the calculated value of the parameter and the reflectance of vitrinite in air R a.
There is a close correlation between and Rа: pair correlation coefficient r = 0.996, determination coefficient – 0.992.
Fig.2.1. Relationship between hard coal parameter and indicator
reflections of vitrinite in air R a (light and dark dots -
various sources)
The presented dependence is described by the equation:
R a \u003d 1.17 - 2.01. (2.6)
Between the calculated value and the reflectance of vitrinite in oil immersion R o. r the connection is non-linear. The research results showed that there is a direct relationship between the structural parameter of vitrinite (Vt) and the indices of liptinite (L) and inertinite (I).
For Kuzbass coals, the relationship between R o. r and the following:
R about. r = 5.493 - 1.3797 + 0.09689 2 . (2.7)
Figure 2.2 shows the relationship between the reflectance of vitrinite in oil immersion Rо. r (op) and calculated by equation (2.7) R o . r(calc).
Fig.2.2. Correlation between experienced R about. r (op) and calculated R o . r (calc)
values of the reflection index of vitrinite coals of Kuzbass
Shown in Fig. 2.2 graphic dependence is characterized by the following statistical indicators: r = 0.990; R 2 \u003d 0.9801.
Thus, the parameter uniquely characterizes the degree of metamorphism hard coal.
2.3. The actual density of coal d r
Is the most important physical characteristic TGI. used
when calculating the porosity of fuels, processes and apparatus for their processing, etc.
The actual density of coal d r is calculated by additivity, taking into account the content in it of the number of moles of carbon, hydrogen, nitrogen, oxygen and sulfur, as well as mineral components according to the equation:
d = V o d + ΣV Mi d Mi + 0.021, (2.8)
where V o and V are the volumetric content of organic matter and individual mineral impurities in coal in fractions of a unit,%;
d and d Mi are the values of the actual densities of the organic matter of coal and mineral impurities;
0.021 - correction factor.
The density of the organic mass of coal is calculated per 100 g of its mass d 100;
d 100 = 100/V 100 , (2.9)
where the value of V 100 is the volumetric content of organic matter in coal, fractions of a unit. Determined by the equation:
V 100 = n C + H n H + N n N + O n O + S n S , (2.10)
where n C o , n H o , n N o , n O o and n S o are the number of moles of carbon, hydrogen, nitrogen and sulfur in 100 g of WMD;
H , N , O and S are empirical coefficients determined experimentally for various coals.
The equation for calculating V 100 of coal vitrinite in the range of carbon content in WMD from 70.5% to 95.0% has the form
V 100 \u003d 5.35 C o + 5.32 H o + 81.61 N o + 4.06 O o + 119.20 S o (2.11)
Figure 2.3 shows a graphical relationship between the calculated and actual values of the density of coal vitrinite, i.e. d = (d)
There is a close correlation between the calculated and experimental values of the true density of vitrinite. In this case, the coefficient of multiple correlation is 0.998, determination - 0.9960.
Fig.2.3. Comparison of calculated and experimental
values of the true density of vitrinite
Yield of volatile substances
Calculated according to the equation:
V daf = V x Vt + V x L + V x I (2.12)
where x Vt ,x L and x I are the proportion of vitrinite, liptinite and inertinite in the composition of coal (x Vt + x L + x I = 1);
V , V and V - dependence of the yield of volatile substances from vitrinite, liptinite and inertinite on the parameter :
V = 63.608 + (2.389 - 0.6527 Vt) Vt , (2.7)
V = 109.344 - 8.439 L , (2.8)
V = 20.23 exp [ (0.4478 – 0.1218 L) ( L – 10.26)], (2.9)
where Vt , L and I are the values of parameters calculated for vitrinite, liptinite and inertinite according to their elemental composition.
Figure 2.4 shows the relationship between the calculated yield of volatile substances on a dry ash-free state and that determined according to GOST. Pair correlation coefficient r = 0.986 and determination R 2 = 0.972.
Fig.2.4. Comparison of experimental V daf (op) and calculated V daf (calc) values
for the release of volatile substances from petrographically inhomogeneous coals
Kuznetsk basin
The relationship of the parameter with the release of volatile substances from coal deposits in South Africa, the USA and Australia is shown in Fig. 2.5.
Fig. 2.5. Dependence of the yield of volatile substances V daf on the structural - chemical
parameters of vitrinite coals:
1 - Kuznetsk coal basin;
2 - coal deposits of South Africa, USA and Australia.
As follows from the data in the figure, the relationship with the release of volatile substances of these countries is very close. The coefficient of pair correlation is 0.969, determination - 0.939. Thus, the parameter with high reliability makes it possible to predict the release of volatile substances from hard coals of world deposits.
Calorific value Q
The most important characteristic TGI as an energy fuel shows the possible amount of heat that is released during the combustion of 1 kg of solid or liquid or 1 m 3 of gaseous fuels.
There are higher (Q S) and lower (Q i) calorific values of fuels.
The gross calorific value is determined in a colorimeter, taking into account the heat of condensation of water vapor formed during the combustion of fuel.
Calculation of heat of combustion solid fuel is produced according to the formula of D.I. Mendeleev based on the data of the elemental composition:
Q = 4.184 [ 81C daf +300H daf +26 (S - O daf)], (2.16)
where Q is the net calorific value, kJ/kg;
4.184 is the conversion factor of kcal to mJ.
The results of TGI studies showed that given the non-identical conditions of coal formation in coal basins, the values of the coefficients for C daf , H daf , S and O daf will be different and the formula for calculating the calorific value has the form:
Q = 4.184, (2.17)
where q C , q H , q SO are coefficients determined experimentally for various coal deposits.
In table. 2.1 shows the regression equations for calculating the net calorific value of coals from various deposits of the TGI of the Russian Federation.
Table 2.1 - Equations for calculating the net calorific value for a coal bomb
various basins of the Russian Federation
The values of the coefficient of pair correlation between the calorific values calculated by the equations and determined by the bomb, presented in the table, show their close correlation. In this case, the coefficient of determination varies within 0.9804 - 0.9880.
The number of fusenized components ∑OK determines the category of hard coal and, in combination with other indicators, makes it possible to assess the use of coal in coking technology.
The parameter ∑OK is the sum of the content of inertinite I and part (2/3) of semivitrinite S v in the coal:
∑OK = I+ 2/3 S v . (2.18)
The research results show that the content of lean components in coals most closely correlates with the combined influence of parameters and H/C. The equation for calculating ∑OK is:
∑OK \u003d b 0 + b 1 + b 2 (H / C) + b 3 (H / C) + b 4 (H / C) 2 + b 5 2. (2.19)
The coefficient of pair correlation of the relationship ∑OC of various grades of coals and charges of the Kuznetsk basin varies from 0.891 to 0.956.
It has been established that there is a higher relationship between the calculated values of ∑OK according to the equations and those determined experimentally for medium metamorphosed coals. The relationship of ∑OK with coals of a higher degree of metamorphism is reduced.
INTRODUCED by Gosstandart of Russia
2. ADOPTED by the Interstate Council for Standardization, Metrology and Certification (Minutes No. 6-94 of October 21, 1994)
|
State name |
Name of the national standardization body |
|
The Republic of Azerbaijan |
Azgosstandart |
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Republic of Armenia |
Armstate standard |
|
Republic of Belarus |
Belgosstandart |
|
Republic of Georgia |
Gruzstandard |
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The Republic of Kazakhstan |
State Standard of the Republic of Kazakhstan |
|
Republic of Kyrgyzstan |
Kyrgyzstandart |
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The Republic of Moldova |
Moldovastandard |
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Russian Federation |
Gosstandart of Russia |
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The Republic of Uzbekistan |
Uzgosstandart |
|
State Standard of Ukraine |
3. This International Standard is the complete authentic text of ISO 7404-5-85 Bituminous and anthracite coal. Methods of petrographic analysis. Part 5. Method for the microscopic determination of vitrinite reflectance indices" and contains additional requirements that reflect the needs of the national economy
4. REPLACE GOST 12113-83
Introduction date 1996-01-01
This International Standard applies to brown coals, hard coals, anthracites, coal mixtures, solid diffuse organics and carbonaceous materials and specifies a method for determining reflectance values.
The vitrinite reflectance index is used to characterize the degree of metamorphism of coals, during their prospecting and exploration, mining and classification, to establish the thermogenetic transformation of solid dispersed organic matter in sedimentary rocks, and also to determine the composition of coal mixtures during enrichment and coking.
Additional requirements reflecting the needs of the national economy are in italics.
1. PURPOSE AND SCOPE
This International Standard specifies a method for determining the minimum, maximum and arbitrary reflectance values using a microscope in immersion oil. and in the air on polished surfaces polished section of briquettes and polished pieces vitrinite component of coal.
GOST 12112-78 Brown coals. Method for determining the petrographic composition
GOST 9414.2-93 Hard coal and anthracite. Methods of petrographic analysis. Part 2. Method for preparing coal samples
3. ESSENCE OF THE METHOD
The essence of the method lies in the measurement and comparison of electric currents arising in a photomultiplier tube (PMT) under the influence of a light flux reflected from the polished surfaces of macerals or submacerals of the test sample and standard samples (etalons) with a set reflection index.
4. SAMPLING AND SAMPLE PREPARATION
4.1. Sampling for the preparation of polished briquettes is carried out according to GOST 10742.
4.2. Polished briquettes are made according to GOST 9414.2.
From the samples intended for measuring the reflection indices with the construction of reflectograms, two polished briquettes with a diameter of at least 20 mm are made.
4.3. For the preparation of polished briquettes from rocks with inclusions of solid dispersed organic matter, the crushed rock is preliminarily enriched, for example, by flotation, by the method of chemical decomposition of the constituent inorganic part of the rocks, and others.
4.4. To prepare polished pieces of coal, samples are taken from the main bed-forming lithotypes with a size of at least 30–30–30 mm. When taking samples from the core of boreholes, it is allowed to take samples with a size of 20 × 20 × 20 mm.
4.5. To prepare polished pieces from rocks with inclusions of solid dispersed organic matter, samples are taken in which inclusions of solid organic matter are microscopically visible or their presence can be assumed by the type of deposits. The size of the samples depends on the possibility of sampling (natural outcrops, mine workings, cores from boreholes).
4.6. The preparation of polished pieces consists of three operations: impregnation in order to give the samples strength and solidity for subsequent grinding and polishing.
4.6.1. Synthetic resins, carnauba wax, rosin with xylene, etc. are used as impregnating agents.
For some types of coals and rocks with inclusions of solid dispersed organic matter, it is sufficient to immerse the sample in the impregnating substance.
If the sample has sufficient strength, the surface perpendicular to the layering plane is lightly ground.
Samples of weakly compacted sandy-clayey rocks containing small scattered organic inclusions are dried in an oven at a temperature of 70 °C for 48 hours before soaking in rosin with xylene.
The samples are tied with wire, to the end of which a label with a passport is attached, and placed in one layer in a porcelain cup, rosin is poured into it, crushed into grains ranging in size from 3 to 7 mm, and xylene is poured (3 cm 3 per 1 g of rosin) so that so that the samples are completely covered with the solution.
Impregnation is carried out in a fume hood when heated on a closed tile for 50 - 60 min until the xylene is completely evaporated. The samples are then removed from the cup and cooled to room temperature.
4.6.2. Grind two mutually parallel planes of the impregnated sample, perpendicular to the layering, and polish one of them.
Grinding and polishing is carried out in accordance with GOST R 50177.2 and GOST 12113.
4.7. In the study of long-term stored polished briquettes and polished pieces, as well as previously measured samples, it is necessary to grind them down by 1.5 - 2 mm before measuring the reflection index and polish them again.
5. MATERIALS AND REAGENTS
5.1. Calibration standards
5.1.1. Reflection index standards, which are samples with a polished surface, meet the following requirements:
a) are isotropic or represent the main section of uniaxial minerals;
b) durable and corrosion resistant;
c) maintain a constant reflectance for a long time;
e) have a low absorption rate.
5.1.2. The standards must be more than 5 mm thick or have the shape trihedral prism (30/60°) to prevent more light from entering the lens than that reflected from its upper (working) surface.
A polished edge is used as a working surface to determine the reflection index. Base and sides of the standard covered with opaque black varnish or placed in a strong opaque frame.
The path of the beam in a wedge-shaped standard inserted into black resin, with photometric measurements reflectance index is shown in Figure 1.
5.1.3. When carrying out measurements, at least three standards are used with reflection indices close to or overlapping the measurement area of the reflection indices of the samples under study. To measure the reflectance of coal equal to 1.0%, standards with reflectances of approximately 0.6 should be used; 1.0; 1.6%.
The average refractive and reflective indices for commonly used standards are shown in Table 1.
5.1.4. The true values of the reflection index of standards are determined in special optical laboratories or calculated from the refractive index.
Knowing the refractive index n and the absorption rate? (if it is significant) of the reference at a wavelength of 546 nm, you can calculate the reflectance ( R) as a percentage according to the formula
If the refractive index is not known, or it is assumed that the surface properties may not accurately correspond to the nominal basic properties, the reflectance is determined by careful comparison with a standard with a known reflectance.
5.1.5. The zero standard is used to eliminate the influence of the dark current of the photomultiplier tube and scattered light in the optical system of the microscope. Optical glass K8 can be used as a zero standard or a polished briquette made of coal with a particle size of less than 0.06 mm and having a depression in the center with a diameter and a depth of 5 mm filled with immersion oil.
Figure 1 - Beam path in a wedge-shaped standard inserted into black resin,
in photometric measurements of the reflectance
Table 1
Average refractive indices of reflection for commonly used standards
5.1.6. When cleaning standards, care must be taken not to damage the polished surface. Otherwise, it is necessary to re-polish its working surface.
5.2. Immersion oil meeting the following requirements:
non-corrosive;
non-drying;
with a refractive index at a wavelength of 546 nm 1.5180 ± 0.0004 at 23 °C;
with temperature coefficient dn/dt less than 0.005 K -1 .
The oil must be free of toxic components and its refractive index must be checked annually.
5.3. Rectified spirit,
5.4. Absorbent cotton wool, fabric for optics.
5.5. Slides and plasticine for fixing the studied samples.
6. EQUIPMENT
6.1. Monocular or a binocular polarizing microscope with a photometer to measure the index in reflected light. The optical parts of the microscope used to measure the reflectance are shown in Figure 2. The constituent parts are not always arranged in the specified sequence.
6.1.1. Light source A. Any light source with stable emission can be used; a 100W quartz halogen lamp is recommended.
6.1.2. Polarizer D- polarizing filter or prism.
6.1.3. Aperture for adjusting light, consisting of two variable apertures, one of which focuses light on the rear focal plane of the lens (illuminator IN), the other - on the surface of the sample (field aperture E). It must be possible to center with respect to the optical axis of the microscope system.
6.1.4. Vertical illuminator - Berek prism, coated plain glass plate or Smith illuminator (combination of mirror with glass plate W). The types of vertical illuminators are shown in Figure 3.
6.1.6. Eyepiece L - two eyepieces, one of which is provided with a crosshair which may be scaled so that the total magnification of the objective, the eyepieces and in some cases the tube is between 250° and 750°. A third eyepiece may be required M on the path of light to the photomultiplier.
A- lamp; B- converging lens IN- aperture of the illuminator; G- thermal filter;
D- polarizer; E- field diaphragm; AND- focusing lens of the field diaphragm;
W- vertical illuminator; AND- lens; R
-
sample; TO- table; L- eyepieces;
M -
third eyepiece; H- measuring aperture, ABOUT- 546 nm interference filter;
P- photomultiplier
Figure 2 - Optical parts of a microscope used to measure the reflectance
6.1.7. A microscope tube having the following attachments:
a) measuring aperture H, which allows you to adjust the light flux reflected into the photomultiplier from the surface of the sample R, area less than 80 microns 2 . The aperture should be centered with the cross hairs of the eyepiece;
b) devices for optical isolation of eyepieces to prevent excess light from entering during measurements;
c) the necessary blackening to absorb scattered light.
NOTE With care, part of the light flux can be diverted to the eyepiece or TV camera for continuous observation when measuring the reflectance.
6.1.8. Filter ABOUT with a bandwidth maximum at (546 ± 5) nm and a bandwidth half-width of less than 30 nm. The filter should be located in the light path directly in front of the photomultiplier.
A- filament; B- converging lens IN -
aperture of the illuminator (position of reflection of the filament);
G- field diaphragm; D- focusing lens of the field diaphragm; E- Berek prism;
AND- reverse focal plane of the lens (the position of the image of the filament and the aperture of the illuminator);
W- lens; AND- sample surface (image position of the field of view);
A- vertical illuminator with Berek prism; b- illuminator with a glass plate; V- Smith's illuminator
Figure 3 - Scheme of vertical illuminators
6.1.9. Photomultiplier P, fixed in a nozzle mounted on a microscope and enabling the light flux through the measuring aperture and the filter to enter the photomultiplier window.
The photomultiplier should be of the type recommended for measuring light fluxes of low intensity, should have sufficient sensitivity at 546 nm and low dark current. Its characteristic should be linear in the measurement region, and the signal should be stable for 2 hours. Usually, a direct multiplier with a diameter of 50 mm is used with an optical input at the end, having 11 diodes.
6.1.10. microscope stage TO, capable of rotating 360° perpendicular to the optical axis, which can be centered by adjusting the stage or lens. The rotating stage is connected to the preparation driver, which ensures the movement of the sample, with a step of 0.5 mm in the directions X And Y, equipped with a device that allows for slight adjustment of movements in both directions within 10 microns.
6.2. DC stabilizer for light source. Characteristics must satisfy the following conditions:
1) lamp power should be 90 - 95% of the norm;
2) fluctuations in lamp power should be less than 0.02% when the power source changes by 10%;
3) ripple at full load less than 0.07%;
4) temperature coefficient less than 0.05% K -1.
6.3. DC voltage stabilizer for photomultiplier.
Characteristics must satisfy the following conditions:
1) voltage fluctuations at the output must be at least 0.05% when the voltage of the current source changes by 10%;
2) ripple at full load less than 0.07%;
3) temperature coefficient less than 0.05% K -1;
4) changing the load from zero to full should not change the output voltage by more than 0.1%.
Note - If during the measurement period the voltage of the power supply drops by 90%, an autotransformer should be installed between the power supply and both stabilizers.
6.4. Indicating device (display), consisting of one of the following devices:
1) a galvanometer with a minimum sensitivity of 10 -10 A/mm;
2) recorder;
3) digital voltmeter or digital indicator.
The instrument shall be adjusted so that its full scale response time is less than 1 s and its resolution is 0.005% reflectance. The device must be equipped with a device for removing the small positive potential that occurs when the photomultiplier is discharged and due to the dark current.
Notes
1. The digital voltmeter or indicator must be able to clearly distinguish the values of the maximum reflectance when the sample is rotated on the stage. Individual values of the reflectance can be stored electronically or recorded on magnetic tape for further processing.
2. A low noise amplifier can be used to amplify the photomultiplier signal when applied to the indicating instrument.
6.5. fixture to give the polished surface of the test sample or reference position parallel to the glass slide (press).
7. MEASUREMENTS
7.1. Equipment preparation (in 7.1.3 and 7.1.4, the letters in parentheses refer to Figure 2).
7.1.1. Initial Operations
Make sure that the room temperature is (23 ± 3) °C.
Include current sources, lights and other electrical equipment. Set the voltage recommended for this photomultiplier by its manufacturer. To stabilize the equipment, it is kept for 30 minutes before the start of measurements.
7.1.2. Microscope adjustment for reflectance measurement.
If an arbitrary reflectance is measured, the polarizer is removed. If the maximum reflectance is measured, the polarizer is set to zero when using a glass plate or Smith illuminator, or at an angle of 45° when using a Berek prism. If a polarizing filter is used, it is checked and replaced if it shows significant discoloration.
7.1.3. Lighting
A drop of immersion oil is applied to the polished surface of the polished briquette mounted on a glass slide and leveled and placed on the microscope stage.
Check the correct adjustment of the microscope for Koehler illumination. Adjust the illuminated field using the field diaphragm ( E) so that its diameter is about 1/3 of the entire field. Illuminator aperture ( IN) are adjusted so as to reduce glare, but without unduly reducing the intensity of the luminous flux. In the future, the size of the adjusted aperture is not changed.
7.1.4. Adjustment of the optical system. Center and focus the image of the field diaphragm. Center the lens ( AND) but relative to the axis of rotation of the object stage and adjust the center of the measuring aperture ( H) so that it coincides either with the cross hairs or with a given point in the field of view of the optical system. If the image of the measuring aperture cannot be seen on the sample, a field containing a small shiny inclusion, such as a pyrite crystal, is selected and aligned with the cross hairs. Adjust the centering of the measuring aperture ( H) until the photomultiplier gives the highest signal.
7.2. Reliability testing and hardware calibration
7.2.1. Hardware stability.
The standard with the highest reflectance is placed under the microscope, focused in immersion oil. The voltage of the photomultiplier is adjusted until the display reading matches the reflectance of the standard (for example, 173 mV corresponds to a reflectance of 173%). The signal must be constant, the change in reading must not exceed 0.02% within 15 minutes.
7.2.2. Changes in readings during rotation of the reflectance standard on the stage.
Place a standard with an oil reflectance of 1.65 to 2.0% on the stage and focus in the immersion oil. Rotate the stage slowly to ensure that the maximum change in readings is less than 2% of the reflectance of the sample taken. If the deviation is higher than this value, it is necessary to check the horizontal position of the standard and ensure its strict perpendicularity to the optical axis and rotation in the same plane. If after this the fluctuations do not become less than 2%, the manufacturer must check the mechanical stability of the stage and the microscope geometry.
7.2.4. Linearity of the photomultiplier signal
Measure the reflectance of the other standards at the same constant voltage and the same light aperture setting to check that the measuring system is linear within the measured limits and that the standards are consistent with their design values. Rotate each standard so that the readings are as close as possible to the calculated value. If the value for any of the standards differs from the calculated reflectance by more than 0.02%, the standard should be cleaned and the calibration process repeated. The standard must be polished again until the reflection index differs from the calculated one by more than 0.02%.
If the reflectance of the standards does not give a linear diagram, check the linearity of the photomultiplier signal using standards from other sources. If they don't give line chart, check the signal linearity again by applying several neutral density calibration filters to reduce the light flux to a known value. If non-linearity of the photomultiplier signal is confirmed, replace the photomultiplier tube and carry out further testing until signal linearity is obtained.
7.2.5. Hardware calibration
Having established the reliability of the apparatus, it is necessary to ensure that the indicating instrument gives the correct readings for the zero standard and the three reflection standards of the test coal, as indicated in 7.2.1 to 7.2.4. The reflectance of each standard shown on the display should not differ from the calculated one by more than 0.02%.
7.3. Vitrinite reflectance measurement
7.3.1. General provisions
The method for measuring the maximum and minimum reflectance values is given in 7.3.2, and for an arbitrary one in 7.3.3. In these subclauses, the term vitrinite refers to one or more submacerals of the vitrinite group.
As discussed in Section 1, the choice of submacerals to be measured determines the result, and therefore it is important to decide which submacerals to measure the reflectance and note them when reporting the results.
7.3.2. Measurement of maximum and minimum vitrinite reflectance in oil.
Install the polarizer and check the apparatus according to 7.1 and 7.2.
Immediately after calibration of the equipment, a leveled polished preparation made from the test sample is placed on a mechanical stage (preparation) that allows measurements to be made starting from one corner. Apply immersion oil to the surface of the sample and focus. Slightly move the specimen with the driver preparation until the cross hairs are focused on a suitable surface of the vitrinite. The surface to be measured must be free from cracks, polishing defects, mineral inclusions or relief and must be at some distance from the boundaries of the maceral.
Light is passed through a photomultiplier and the table is rotated 360° at a speed of not more than 10 min -1 . Record the largest and smallest values of the reflection index, which is noted during the rotation of the table.
NOTE When the slide is rotated 360°, ideally, two identical maximum and minimum readings can be obtained. If the two readings are very different, the cause should be determined and the error corrected. Sometimes the cause of the error can be air bubbles in the oil getting into the measured area. In this case, the readings are ignored and air bubbles are eliminated by lowering or raising the microscope stage (depending on the design). The front surface of the objective lens is wiped with an optical cloth, a drop of oil is again applied to the surface of the sample and focusing is performed.
The sample is moved in the direction X(step length 0.5 mm) and take measurements when the crosshairs hit a suitable surface of the vitrinite. In order to be sure that the measurements are made on a suitable site of the vitrinite, the sample can be moved by the slider up to 10 µm. At the end of the path, the sample moves to the next line: the distance between the lines is at least 0.5 mm. The distance between the lines is chosen so that the measurements are distributed evenly on the surface of the section. Continue to measure the reflectance using this testing procedure.
Every 60 min, recheck the calibration of the apparatus against the standard closest to the highest reflectance (7.2.5). If the reflectance of the standard differs by more than 0.01% from the theoretical value, discard the last reading and perform them again after recalibrating the apparatus against all standards.
Reflectance measurements are made until the required number of measurements is obtained. If the polished briquette is prepared from coal of one layer, then from 40 to 100 measurements and more are made (see table 3 ). The number of measurements increases with the degree of vitrinite anisotropy. In each measured grain, the maximum and minimum values of the count are determined and during the rotation of the microscope stage. The average maximum and minimum reflectance values are calculated as the arithmetic mean of the maximum and minimum reports.
If the sample used is a mixture of coals, then 500 measurements are made.
On each polished specimen, 10 or more vitrinite areas should be measured, depending on the degree of anisotropy of the test sample and the objectives of the study.
Before starting measurements, the polished specimen is set so that the layering plane is perpendicular to the incident beam of the optical system of the microscope. At each measured point, the position of the maximum reading is found, and then readings are recorded every 90° of the microscope stage rotation when it is rotated 360°.
Maximum and minimum reflectance (R 0,max and R 0, min) calculated as the arithmetic mean of the maximum and minimum readings, respectively.
7.3.3. Measurement of an arbitrary vitrinite reflectance in immersion oil (R 0, r)
Use the procedure described in 7.3.2, but without polarizer and sample rotation. Perform calibration as described in 7.2.5
Measure the vitrinite reflectance until the required number of measurements is recorded.
On each polished briquette, it is necessary to perform from 40 to 100 or more measurements (table 3 ) depending on the homogeneity and degree of anisotropy of the test sample.
The number of measurements increases with an increase in heterogeneity in the composition of the huminite and vitrinite group, as well as with a pronounced anisotropy of hard coals and anthracites.
The number of measurements for samples containing solid dispersed organic matter is determined by the nature and size of these inclusions and can be significantly lower.
To establish the composition of coal mixtures from reflectograms, it is necessary to carry out at least 500 measurements on two samples of the coal sample under study. If the participation of coals of various degrees of metamorphism, which are part of the charge, cannot be established unambiguously, another 100 measurements are carried out and in the future until their number is sufficient. Limit number of measurements - 1000.
On each polished piece, up to 20 measurements are performed in two mutually perpendicular directions. To do this, the polished piece is set so that the layering plane is perpendicular to the incident beam of the optical system of the microscope. The sites for measurements are chosen so that they are evenly distributed over the entire surface of the vitrinite of the studied polished specimen.
Arbitrary reflection index (R 0, r ) is calculated as the arithmetic mean of all measurements.
7.3.4. Reflection measurements in air.
Definitions of the maximum, minimum and arbitrary reflection indices (R a, max , Ra, min And R a, r) may be carried out for a preliminary assessment of the stages of metamorphism.
Measurements in air are carried out similarly to measurements in immersion oil at lower values of aperture stop, illuminator voltage, and PMT operating voltage.
On the studied polished briquette, it is necessary to perform 20 - 30 measurements, polished - 10 or more.
8. PROCESSING THE RESULTS
8.1. Results can be expressed as a single value or as a series of numbers in 0.05% reflectance intervals (1 / 2 V-step) or at intervals of 0.10% of the reflection index ( V-step). The average reflectance and standard deviation are calculated as follows:
1) If individual readings are known, then the average reflectance and standard deviation are calculated using formulas (1) and (2), respectively:
(2)
Where ?R- average maximum, average minimum or average arbitrary reflection index, %.
Ri- individual indication (measurement);
n- number of measurements;
Standard deviation.
2) If the results are presented as a series of measurements in 1 / 2 V-step or V-step, use the following equations:
Where R t- average value 1 / 2 V-step or V-step;
X- number of reflectance measurements in 1 / 2 V-step or V-step.
Register vitrinite submacerals, which include values ?R no matter which reflectance was measured, the maximum, minimum or arbitrary, and the number of measurement points. Percentage of vitrinite for each 1/2 V-step or V-step can be represented as a reflectogram. An example of expressing the results is given in Table 2, the corresponding reflectogram is in Figure 4.
Note - V-step has a range of 0.1 reflectance, and 1/2 has a range of 0.05%. To avoid overlapping reflectance values expressed to the second decimal place, the ranges of values are presented, for example, as follows:
V- step - 0.60 - 0.69; 0.70 - 0.79 etc. (incl.).
1 / 2 V- steps: 0.60 - 0.64; 0.65 - 0.69 etc. (incl.).
The average value of the series (0.60 - 0.69) is 0.645.
The average value of the series (0.60 - 0.64) is 0.62.
8.2. Optionally, an arbitrary reflection index (R 0, r ) is calculated from the average values of the maximum and minimum reflectance values according to the formulas:
for polished ore R 0, r = 2 / 3 R 0, max + 1 / 3 R 0, min
for polished briquette
Value occupies an intermediate position between R 0, max and R 0, min And associated with the grain orientation in the polished briquette.
8.3. As an additional parameter, the reflection anisotropy index (AR) is calculated using the formulas:
8.4. The processing of measurement results in ordinary and polarized light in air on polished briquettes and polished pieces is carried out similarly to the processing of measurement results in immersion oil (8.1 ).
Figure 4 - Reflectogram compiled according to the results of table 2
table 2
Measured reflectance arbitrary
Submacerals of vitrinitis telocollinitis and desmocollinitis
|
Reflection index |
Number of observations |
Percentage of Observations |
Total number of measurements n = 500
Average reflectance ?R 0, r = 1.32%
Standard deviation? = 0.20%
9. PRECISION
9.1. Convergence
Convergence of the definitions of the mean values of the maximum, minimum or arbitrary reflectance is the value by which two separate readings differ, taken with the same number of measurements by the same operator on the same slide using the same apparatus at confidence level 95 %.
Convergence is calculated by the formula
Where? t- theoretical standard deviation.
Convergence depends on a number of factors including:
1) limited calibration accuracy with reflectance standards (6.2.5);
2) allowable calibration drift during measurements (6.3.2);
3) the number of measurements made and the range of values of the reflectance index for vitrinite of one coal seam.
The overall effect of these factors can be expressed as a standard deviation of the average reflectance of up to 0.02% for a sample of one individual coal from one seam. This corresponds to a convergence of up to 0.06%.
9.2. Reproducibility
The reproducibility of determinations of the average values of the maximum, minimum or arbitrary indicators is the value by which the values of two determinations performed with the same number of measurements by two different operators differ on two different preparations made from the same sample and using different equipment, with a confidence probability 95%.
Reproducibility is calculated by the formula
Where? 0 is the actual standard deviation.
If operators are adequately trained to identify vitrinite or the corresponding submacerals, and the reflectance of the reference is reliably known, the standard deviations of mean reflectance determinations by different operators in different laboratories are 0.03%. The reproducibility is thus 0.08%
9.3. Permissible discrepancies between the results of the average values of the reflection indicators of the two definitions are indicated in the table 3 .
Table 3
|
Reflection index, % |
Permissible discrepancies % abs. |
Number of measurements |
|
|
in one laboratory |
in different laboratories |
||
|
Up to 1.0 incl. |
|||
10. TEST REPORT
The test report must include:
2) all details necessary to identify the sample;
3) total number of measurements;
4) the type of measurements made, i.e. maximum, minimum or an arbitrary reflection index;
5) the type and ratio of vitrinite submacerals used in this definition;
6) the results obtained;
7) other features of the sample noticed during the analysis and which may be useful in the use of the results.
