Decree of the Ministry of Health No. 180 / 2002 Coll.
Decree of the Ministry of Health amending Decree No. 1 / 1998 Coll., laying down requirements on the quality, procedure for preparation, testing, storage and dosing of medicinal products (Czech Pharmacopoeia 1997), as amended
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180
DECLARATION
Ministry of Health
of 22 April 2002
amending Decree No. 1 / 1998 of the Ministry of Health Coll., laying down quality requirements, procedure for the preparation, testing, storage and dosage of medicinal products (Czech Pharmacopoeia 1997), as amended
The Ministry of Health, after consulting the Ministry of Agriculture and the Ministry of Industry and Trade, provides, pursuant to § 75 (4) of Act No. 79 / 1997 Coll., on Medicines and on amendments and additions to certain related laws, as amended by Act No. 149 / 2000 Coll.:
Decree No. 1 / 1998 Coll., laying down quality requirements, procedure for the preparation, testing, storage and dosing of medicinal products (Czech Pharmacopoeia 1997), as amended by Decree No. 296 / 1999 Coll. and Decree No. 48 / 2001 Coll., is amended as follows:
1. In Annex Part 2, the test methods, Chapter 2.1. Instruments and other test aids, Chapter 2.1.5 and Chapter 2.1.6 are as follows:
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2.1.5 Test tubes for comparative tests
The test tubes for comparative tests are colourless glass tubes with a uniform internal diameter, the bottom of which is flat and transparent.
The column of the liquid shall be observed in the diffuse light from the top in the direction of the longitudinal axis of the test tube against white or black background if necessary.
The use of test tubes with an inside diameter of 16 mm is envisaged. Test tubes with a larger internal diameter may also be used provided that the volume of the test liquid increases so that the column height in the test tube is not less than the column height of the prescribed volume in the test tube with an internal diameter of 16 mm.
2.1.6 Detection tubes for gases
Detection tubes for gases are sealed tubes made of transparent inert material, which allow gas to pass after breaking the fences. They contain probers adsorbed on inert media that are suitable for visual detection of the monitored substance. If necessary, they may also contain pressurised chemical filters to remove substances that interfere with detection of the substance. The gas detection tube contains either one test tube for the detection of a given substance or more probers for the detection of several different substances (single layer or multilayer tube).
The test shall be performed by passing through the prescribed volume of the test gas through the detection tube. The length of the coloured layer or the intensity of the colour change indicates the presence of the monitored substances on the divided scale and indicates their approximate gas content.
Calibration of detection tubes shall be verified according to the manufacturer's instructions.
Working conditions. The test shall be carried out according to the manufacturer's instructions or the following procedure.
The gas source shall be connected to the appropriate pressure regulator and needle valve. The pivot end with Y shall be connected to the needle valve and the flow rate of the test gas adjusted to pass through the tube at an appropriate speed, see Figure 2.1.6-1. The detection tube shall be prepared and connected to the measuring pump as instructed by the manufacturer. The free end of the detection tube shall be connected by a short hose to the other end of the Y-part. The pump shall be used to draw the necessary amount of gas so that the detection tube passes through the appropriate volume of test gas. Subtract the value given by the length of the coloured layer or by the intensity of discoloration on the divided scale. In the event of a negative result, detection tubes may be verified using a calibration gas containing the monitored substance. Due to the wide range of compressor oils used, it is necessary to verify the reactivity of the detection tube for the oil used. Information on the reactivity of different oils is described in the leaflet supplied at the same time as the tube. If the oil used is not specified in the leaflet, the tube manufacturer shall verify the reactivity of the tube and, if necessary, identify the tube specific to that oil.
Carbon dioxide detector tube. It is a glass sealed tube containing adsorption filters and suitable carriers for hydrazine and crystal violet as detection probers. The minimum detectable value is 100 ml / m3 with a relative standard deviation of not more than ± 15%.
Sulfur oxide detection tube. There is a glass sealed tube containing adsorption filters and suitable holders for iodine and starch as detection probers. The minimum detectable value is 0,5 ml / m3 with a relative standard deviation of not more than ± 15%.
Oil detection tube. It is a glass sealed tube containing adsorption filters and suitable carriers for sulphuric acid as a detection probe. The minimum detectable value is 0,1 mg / m3 with a relative standard deviation of not more than ± 30%.
Nitric oxide and nitrogen dioxide detection tube. It is a glass sealed tube containing adsorption filters and suitable carriers for the oxidation layer (hexavalent chromium salt) and diphenylbenzidine as detection probers. The minimum detectable value is 0,5 ml / m3 with a relative standard deviation of not more than ± 15%.
Carbon monoxide detector tube. It is a glass sealed tube containing adsorption filters and suitable carriers for iodide oxide, selenium oxide and sulphuric acid as detection probers. The minimum detectable value is 5 ml / m3 (or less) with a relative standard deviation of not more than ± 15%.
Hydrogen sulphide detection tube. It is a glass sealed tube containing adsorption filters and suitable support for lead salt as a detection test. The minimum detectable value is 1 ml / m3 (or less) with a relative standard deviation of not more than ± 10%.
Water vapour detection tube. It is a glass sealed tube containing adsorption filters and suitable carriers for magnesium perchlorate as a detection probe. The minimum detectable value is 67 ml / m3 (or less) with a relative standard deviation of not more than ± 20%.
Fig. 2.1.6-1 Diagram of detection tube for gases
1. source of gas,
2. pressure regulator,
3. needle valve,
4. coupling part Y,
5. detection tube,
6. a pump for pumping gas through a tube,
7th output into the atmosphere.
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2. In Part 2 of the Annex to the Test Method, Chapter 2.2. Physical and physico-chemical methods, Chapter 2.2.4:
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2.2.4. Relationship between the reaction of the solution, approximate pH values and discoloration of some indicators
Add 0,1 ml of indicator to the 10 ml of the test solution unless prescribed in Table 2.2.4-1 otherwise.
Tab. 2.2.4-1
| Reakce | PH | Indikátor | Zbarvení |
|---|---|---|---|
| zásaditá | > 8 | papír lakmusový červený R | modré |
| modř thymolová RS (0,05 ml) | šedé nebo fialově modré | ||
| slabě zásaditá | 8,0 -10,0 | fenolftalein RS (0,05 ml) | bezbarvé nebo růžové |
| modř thymolová RS (0,05 ml) | šedé | ||
| silně zásaditá | > 10 | papír s fenolftaleinem R | červené |
| modř thymolová RS (0,05 ml) | fialově modré | ||
| neutrální | 6,0 - 8,0 | červeň methylová RS | žluté |
| červeň fenolová RS (0,05 ml) | |||
| neutrální na červeň | 4,5 - 6,0 | červeň methylová RS | oranžově červené |
| methylovou neutrální na fenolftalein | < 8,0 | fenolftalein RS (0,05 ml) | bezbarvé; růžové nebo červené po přidání 0,05 ml zásady 0,1 mol/l |
| kyselá | < 6 | červeň methylová RS | oranžové nebo červené |
| modř bromthymolová RS1 | žluté | ||
| slabě kyselá | 4,0 - 6,0 | červeň methylová RS | oranžové |
| zeleň bromkresolová RS | zelené nebo modré | ||
| silně kyselá | < 4 | papír s červení Kongo R | zelené nebo modré |
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3. In Part 2 of the Annex to the Test Method, Chapter 2.2. Physical and physico-chemical methods, Chapter 2.2.27 to Chapter 2.2.30 read:
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2.2.27 Thin-layer chromatography
Thin-layer chromatography is a separation method for which the stationary phase forms an appropriate material applied in a uniform thin layer to a glass, metal or plastic base (plate). Determine solutions are applied to the layer before development. The separation is based on adsorption, distribution, ion exchange or a combination of these mechanisms and is caused by migration (development) of dissolved substances in the solvent or an appropriate mixture of solvents (mobile phase) with a thin layer (stationary phase).
Equipment
Plates. Chromatography shall be carried out using pre-coated plates, as described in the statum of the Examiner (4.1.1).
Preliminary treatment of layers. Before splitting, the layers, if necessary, may be washed, e.g. by developing an appropriate solvent. The layers may also be impregnated by development, immersion or spraying. If necessary, the layers can be activated by heating at 100 ° C to 105 ° C for 1 h before use.
Chromatographic chamber with flat or double gouge bottom made of inert transparent material of suitable size for used plates, equipped with a well-sealed lid. The horizontal development chamber shall be equipped with a channel for the mobile phase and an additional device enabling direct contact between the mobile phase and the stationary phase.
Micropipettes, microsyringes (injection), calibrated capillaries for one use, do not fight the application device, which is suitable for proper application of solutions.
Fluorescent detection equipment for direct fluorescence or fluorescence extinguishing.
Detection agents for the detection of isolated spots by spraying, exposure to vapour or immersion.
Working procedure
Vertical evolution. The walls of the chromatographic chamber shall be unloaded with filter paper. The layer is poured into the chromatographic chamber according to the size of the mobile phase so that, after impregnation of the filter paper, the layer is immersed in a suitable depth, due to the dimensions of the plate used. For saturation, the chamber is closed with a lid and left to stand at 20 ° C to 25 ° C for 1 hour. Unless otherwise prescribed, chromatographic division shall be carried out in a saturated chamber.
The prescribed volume of solutions shall be applied after sufficient small doses in the form of strips or circular spots at an appropriate distance from the lower edge and side edges of the plate. The solutions shall be applied to the start parallel to the lower edge of the plate so that the distance between the stains is at least 10 mm.
After evaporation of the solvent from the applied solutions, the layer plate shall be placed in a chromatographic chamber so that it is in the most vertical position and the spots or strips are above the level of the mobile phase. The chamber shall be closed, maintained at 20 ° C to 25 ° C and protected from sunlight. The layer plate shall be removed when the mobile phase reaches the prescribed distance, dried and degraded in the prescribed manner.
For two-dimensional chromatography, the layer is dried after the first development and the second development is carried out in a direction perpendicular to the first development direction.
Horizontal development. The prescribed volume of solutions after small enough doses shall be applied to give rise to circular spots of a diameter of 1 mm to 2 mm or strips 5 mm to 10 mm times 1 mm to 2 mm at an appropriate distance from the lower edge and side edges of the plate. The solutions shall be applied to the start parallel to the lower edge of the plate so that the distance between the stains is at least 5 mm. After evaporation of the solvent from the applied solutions, the assistance of the syringe or pipette shall be introduced into the gill of the developing chamber by the appropriate amount of the mobile phase, the plate with a layer placed horizontally in the chromatographic chamber and connected with the mobile phase by the device as instructed by the manufacturer. If prescribed, the development shall begin simultaneously from both sides. The chamber shall be closed and maintained at 20 ° C to 25 ° C. The layer plate shall be removed when the mobile phase reaches the distance prescribed in the article, dried and chromatograms degraded in the prescribed manner.
For two-dimensional chromatography, the layer is dried after the first development and the second development is carried out in a direction perpendicular to the first development direction.
Visual evaluation
Identity test. The main stain on the chromatogram of the test solution is visually compared to the corresponding stain on the chromatogram of the comparison solution by comparing the colour, size and retention factor (RF) of both spots. The retention factor (RF) is defined as the ratio of the distance of the centre of the stain to the distance of the front of the mobile phase measured from the application point.
Verification of separation capability for identity tests. Usually it is sufficient to carry out the aptitude test described in the statum of the Examiner (411). Only in specific cases may an additional criterion for the eligibility test be prescribed in the article.
Test for related substances. The secondary stain (s) on the chromatogram of the test solution shall be visually compared (s) with the corresponding stain (s) on the chromatogram of the comparison solution containing impurity (s) or with the stain on the chromatogram of the comparison solution prepared by dilution of the test solution.
Verification of separation capability. The requirements for verification of separation capability are set out in the relevant Article.
Verification of detection capability. The detection capability is sufficient if the stain (or strip) is clearly visible on the chromatogram of the most diluted comparative solution.
Quantitative determination
The distinction and division requirements are set out in the various articles.
Substances separated by thin-film chromatography and detachable in ultraviolet or visible spectrum areas may be determined directly on the plate with a layer using suitable instrumentation. The layer plate shall be evaluated by measuring the reflectance or transmittance of the falling light, moving either the plate or the measuring device. Similarly, fluorescence may be measured using an appropriate optical system. Substances containing radionuclides may be quantified in three ways either by direct measurement of radioactivity along the whole chromatogram (see Radiopharmaca) or by cutting the plate into strips by measuring the radioactivity of each single strip using a suitable detector and / or by scraping a stationary phase and dissolving it into an appropriate scintillation fluid by measuring its radioactivity using a liquid scintillation detector.
Device. The instrumentation for direct measurement on the plate shall consist of:
- equipment for precise positioning and reproducible dosing of substances per layer,
- a mechanical device for moving the plate or measuring device in the direction of the x-axis or the y-axis,
- a recorder and a suitable integrator or computer,
- for substances which are detachable in ultraviolet or visible spectrum areas for measuring reflectance or transmittance, a photometer with a light source, an optical device capable of generating monochrome light and a photoelectric cell corresponding to sensitivity, for measuring fluorescence in addition to a monochrome filter for displacing a particular spectral area of emitted radiation,
- for substances containing radionuclides, an appropriate device for measuring radioactivity for which the verified linearity area is.
Procedure. In the prescribed manner, prepare the test solution and, if necessary, prepare the comparator solutions of the determined substance in the same solvent as the test solution. The same volumes of all prepared solutions shall be applied and the chromatogram developed.
Substances which are detachable in ultraviolet and visible spectrum areas. Prepare and apply at least three comparative solutions in which the concentrations of the test substance are within the range of its expected content in the test solution (about 80% 100% and 120%). After completion of development, spray the prescribed reagent if necessary and record reflectance, transmittance or fluorescence of the chromatograms of the test solution and comparison solutions. The measured values are used to calculate the amount of substance in the test solution.
Substances containing radionuclides. Prepare and apply a test solution with a concentration of about 100% of the expected value. The radioactivity shall be determined as a function of the distance and radioactivity of each resulting pike and expressed as a percentage of the total amount of radioactivity.
The criteria for assessing the capability of the system are described in the state Chromatographic separation method (2.2.46). The extent to which individual parameters of the chromatographic system can be moved to meet the eligibility criteria of the system is also indicated in this state.
2.2.28 Gas chromatography
Gas chromatography (GC) is a separation method based on the difference in the distribution of substances between two non-mixing phases, the mobile phase is carrier gas, moving through or along the stationary phase, which is placed in the column. It is applicable to substances or their derivatives which can be converted into gaseous phase at the temperatures used.
Gas chromatography is based on a mechanism of adsorption, distribution or elimination.
Device
The apparatus consists of a dosing device, a chromatographic column located in a thermostat, a detector and a data processing system (or an integrator, or a recorder). The carrier gas flows through the column of controlled speed or at controlled pressure into the detector.
The determination shall be made either at a constant temperature or according to a given temperature programme.
Dosing device
Direct injection of solutions is the usual way of dosing, unless otherwise specified in the article. The injection may be done either directly at the beginning of the column using a syringe or a dosing loop, or in a injection chamber which may be provided with a flow divider.
Injections of the gaseous phase may be performed using static or dynamic headspace dosing systems.
Dynamic headspace dosing systems for adsorption and desorption contain a bubbling device by which volatile substances are released from the solution and washed into an adsorption column maintained at low temperature. The retained substances are then desorbed into the mobile phase by rapidly heating the adsorption box.
Static headspace dosing systems shall contain a thermostatic chamber in which closed containers are inserted, containing solid or liquid samples for a pre-determined period, necessary to balance the volatile components of the sample between the solid or liquid phase and the gaseous phase. Once this balance has been achieved, a predetermined amount of the gas phase from the vial is given into the gas chromatograph.
Stationary Phase
Stationary phases are located in columns which may be:
- quartz capillary columns on the walls of which stationary phases are applied,
- columns filled with inert particles impregnated with a stationary phase,
- columns filled with fixed stationary phases.
Capillary columns have internal diameter 0,1 mm to 0,53 mm and length 5 m to 60 m. The liquid stationary phase, which may be chemically bound to the inner surface of the column, consists of a film of 0,1 μm to 5,0 μm thick.
Filling columns made of glass or metal are usually 1 m to 3 m long with an internal diameter of 2 mm to 4 mm. Stationary phases are most commonly porous polymers or solid carriers impregnated with liquid phase.
Carriers for the analysis of polar substances in columns with stationary stages of low polarity and low coverage must be inert in order to avoid spades. The reactivity of the materials from which the carrier is made may be reduced by silanisation, which prevents their coverage by the liquid phase. Acid washed hot calcined diatomite quartz is often used. The carriers have different particle sizes. The most commonly used are particles between 150 μm and 180 μm and 125 μm to 150 μm.
Mobile Phase
The retention time of the component and the efficiency of the column depend on the flow rate of the carrier gas, the retention time is directly proportional to the column length and the resolution is proportional to the square root of the column length. For load columns, the flow rate of the carrier gas shall normally be expressed in millilitres per minute at atmospheric pressure and room temperature and measured at the output of the detector, either by calibrated mechanical device or by a bubble flow meter, while the column is thermostatised to working temperature. The linear flow rate of the carrier gas by the filling column is indirectly proportional to the square root of the internal diameter of the column at a given volume flow rate. The flow rates of 60 ml / min in a column with an internal diameter of 4 mm and 15 ml / min in a column with an internal diameter of 2 mm give the same linear speeds and therefore similar retention times.
The most frequently used load-cell carrier gases are helium or nitrogen, while for capillary columns nitrogen, helium and hydrogen.
Detectors
Flame ionisation detectors are usually used, but according to the purpose of the analysis, other electron capture detectors of nitrogen-phosphorus, mass spectrometer, thermal conductivity, infrared spectrophotometer with Fourier transformation and others can be used.
Working procedure
The column, dosing equipment and detector shall be stabilised at the temperatures and flows of the gases prescribed in the pharmacopoeia cells unless a stable zero line is reached. Prepare the test substance (s) solution (s) and compare solution (s) as prescribed. The solutions shall be free of particles.
The criteria for assessing the suitability of the system are described in the state Chromatographic separation method (2.2.46). This status also indicates the extent to which the parameters of the chromatographic system can be set to meet the requirements of the aptitude test.
Static head-space gas chromatography
Headspace gas chromatography is a method particularly suitable for dividing and determining volatile substances present in solid or liquid samples. This method is based on a gas phase analysis that is balanced with the solid or liquid phase.
Device
The apparatus shall consist of a gas chromatograph equipped with a device for retracting the test sample which may be connected to a module which automatically controls pressure and temperature. If necessary, a solvent elimination device may be attached.
The analysed sample shall be placed in a vial fitted with an appropriate cap and a valve system allowing the passage of the carrier gas. The vial shall be placed in a thermostatic container heated to an appropriate temperature according to the nature of the sample being tested.
The sample shall be heated to this temperature long enough to establish a balance between the solid or liquid phase and the gaseous phase.
The carrier gas is sealed into the container and after the prescribed time a suitable valve is opened so that the gas expands and takes the gaseous substances into the chromatographic column.
It is also possible to use a gas-tight syringe and a regular chromatograph instead of a chromatograph specially equipped for sealing the samples. The balance is then established in a separate chamber and the gaseous phase is dosed into the column while ensuring conditions ensuring that the balance is not impaired.
Working procedure
The use of comparative samples shall determine appropriate instrumental conditions to achieve an appropriate response.
Direct calibration
A separate test sample and all comparative samples, as prescribed in the Pharmacopoeia, shall be placed in the same bottles, avoiding contact between the dosing device and the samples.
The bottles shall be sealed hermetically and placed in a thermostatic space maintained at the temperature and pressure prescribed in the article, and a chromatographic analysis shall be carried out under the prescribed conditions when equilibrium is established.
Standard addition
The same volumes of the test sample shall be placed in a set of the same suitable vials. Appropriate quantities of comparative samples containing known concentrations of the determined substance shall be added to all vials, except one, so that a series of samples containing gradually increasing concentrations of the determined substance is produced.
The bottles shall be sealed hermetically and placed in a thermostatic space maintained at the temperature and pressure prescribed in the pharmacopoeia. A chromatographic analysis under prescribed conditions shall be carried out once the balance has been established.
The method of least squares shall calculate the linear equation of the line and determine the concentration of the determined substance in the test sample.
Alternatively, the mean value of measured data against the added amounts of the determined substance shall be drawn into the graph. The line joining the points on the graph is extrapolated until it crosses the concentration axis. The distance between this point and the intersection of the axes is the concentration of the determined substance in the test sample.
Sequential sampling (repeated head-space extraction)
Where the method of gradual collection is required, it is detailed in the Pharmacopoeia.
2.2.29 Liquid chromatography
Liquid chromatography (LC) is a separate method based on the difference in the distribution of substances between two non-mixed phases, of which the mobile phase is a liquid that passes through a stationary phase filled into a column.
The LC is mainly based on a mechanism of adsorption, distribution, ion exchange, excretion or stereochemical interactions.
Device
The apparatus consists of a pump system, a dosing device, a chromatographic column (a column temperature regulator may also be used), a detector and a data processing device (or an integrator or recorder). The mobile phase is brought into the system from one or more storage tanks and usually flows through a constant speed column and sweat through a detector.
Pumps systems
LC pumping systems are needed for bringing mobile phase constant flow rate. The pressure fluctuation should be as low as possible, which is achieved by, for example, passing the pressurised solvent through the pulses control device. The hoses and all connections shall be able to withstand the pressure developed by the pumping system. LC pumping systems may be equipped with a device to remove released air bubbles.
Microprocessor-controlled systems are able to accurately deliver the mobile phase either with constant composition (isocratic elution) or with a composition that changes according to a pre-specified program (gradient elution). In the case of gradion elution, pumping systems are used which deliver solvent or solvents from multiple containers and the mixing of solvents is achieved either before pressurisation or in the pressurised part of the pump or pumps.
Dosing device
The sample solution is dosed into or near the upper end of the column by means of a dosing device capable of operating at high pressure. Dosing loop devices of constant or variable volume are used for hand or automatic dispensers. Manual dosing with partially filled loop can cause lower accuracy of the injected volume.
Stationary Phase
The LC uses many types of stationary phases such as:
- silica gel aluminium oxide or porous graphite used in chromatography with normal phases where separation is based on differences in adsorption and / or distribution,
- resins or polymers with acidic or basic groups used in ion exchange chromatography, where separation is based on competition between ions to be separated and ions contained in the mobile phase,
- porous silica gel or polymers used in elimination chromatography where separation is based on differences in molecular volumes,
- various types of chemically modified carriers prepared from polymers of silica gel or porous graphite used in reverse-phase chromatography where separation is mainly based on the distribution of molecules between mobile and stationary phases,
- special chemically modified stationary phases such as cellulose or amylose derivatives, proteins or cyclodextrin peptides, etc., used for the separation of enantiomers (chiral chromatography).
Most separation is based on a distribution mechanism and uses chemically modified silica gel as a stationary phase and polar solvent as a mobile phase. The surface of the carrier, such as the silanol group of silica gel, is adjusted to react with various silanisation agents to form covalently bound silyl derivatives that cover the variable amount of active places on the carrier. The nature of the coupled phase is an important parameter for determining the separation properties of the chromatographic system.
Commonly used bound phases are listed below:
| oktyl | = Si-(CH2)7-CH3 | C8 |
| oktadecyl | = Si-(CH2)17-CH3 | C18 |
| fenyl | = Si-(CH2)n-OC6H5) | C6H5 |
| kyanpropyl | = Si-(CH2)3-CN | CN |
| aminopropyl | = Si-(CH2)3-NH2 | NH2 |
| diol | = Si-(CH2)3-OCH(OH) CH2-OH |
Unless otherwise stated by the manufacturer, reverse phase chromatography columns with silica gel-based load are expected to be stable in mobile phases with apparent pH 2,0 to 8 0. Colons filled with porous graphite or particles of polymer materials such as styrene divinylbenzene, copolymer, are stable in the wider pH range.
In some cases, normal phase chromatography is appropriate, which uses unmodified silica gel, porous graphite or polar groups chemically modified silica gel as a stationary phase, e.g. with cyanide or diol groups in combination with non-polar mobile phase.
For analytical separation, stationary phases of 3 μm to 10 μm are commonly used. Particles may have spherical or irregular shapes, various porosity and specific surfaces. These parameters contribute to the chromatographic behaviour of individual stationary phases. In the case of reversed phases, additional characteristics of the stationary phase are its type, the degree of binding expressed, for example, as the bound carbon content, an indication of the possible removal of surface silanol groups. If the stationary phase contains residual surface silanol groups, the analysed substances, in particular basic, may show spades.
The analytical chromatography uses columns made of stainless steel, unless otherwise specified in the article. They have different lengths and internal diameters. Colons with an internal diameter of less than 2 mm are often referred to as microcolumns. The constant temperature of the mobile phase and column shall be maintained during the analysis. Most separation is performed at room temperature, but columns can also be heated to a higher temperature to achieve higher efficiency. It is recommended that the columns are not heated to a temperature above 60 ° C due to the risk of degradation of the stationary phase or changes in the composition of the mobile phase.
Mobile Phase
Less polar mobile phases are used in chromatography with normal phases. In order to achieve reproducible results, it is necessary to closely monitor the presence of water in the mobile phase. The reverse phase chromatography uses aqueous mobile phases with or without organic solvent.
Mobile phase components are usually filtered to remove particles greater than 0,45 μm. Multi-component mobile phases are prepared by measuring the required volumes (if no weight is prescribed) of individual components and by mixing them. Another option is to deliver solvents using individual valves controlled pumps that allow mixing of components in the desired ratio. The solvent is usually degasified by the bubbling of helium in the ultrasound bath before pumping into the system, or membrane or vacuum equipment is used directly to prevent the formation of bubbles in the detector.
Solutions for the preparation of the mobile phase generally do not contain stabilisers and are permeable to the wavelength used for detection in the ultraviolet spectrum area. The solvents used and other components of the mobile phase should be of suitable quality. If pH adjustment is necessary, it is performed only with the aqueous part of the mobile phase, not with the whole mixture. If silencing solutions are used, the system shall be properly washed after chromatographic analysis of the mixture of water and organic solvent used in the mobile phase (5% (V / V)) to prevent the crystallization of salts.
Mobile phases may contain other components, such as antiions for ion pair chromatography or chiral selector for achial stationary phase chromatography.
Detectors
The most commonly used detectors are spectrophotometers for ultraviolet and visible measurements (UV / Vis) including diode field detectors. Fluorescent spectrophotometers, differential refractometers, electrochemical detectors, mass spectrometers, light dispersion detectors or radioactivity measurements and other special detectors may also be used.
Working procedure
With the prescribed mobile phase and flow rate, the column shall be balanced at room temperature or temperature as specified in the pharmacopoeia at which the baseline stability is achieved.
Prepare the solution (s) of the test substance and compare solution (s). The solutions must not contain solid particles.
Criteria for assessing the suitability of the system are described in the status of Chromatographic separation method (2.2.46). This status also shows the extent to which the parameters of the chromatographic system can be set to meet the requirements of the aptitude test.
2.2.30 Excretion chromatography
Elimination chromatography is a separation method that divides molecules according to their size. If an organic mobile phase is used, the method is known as gel chromatography, if an aqueous mobile phase is called the gel filtration method.
The sample shall be applied to a column filled with gel or filled with porous particles and carried by a mobile phase. The division according to the molecular size occurs by repeated replacement of molecules of dissolved substances between the solvent in the mobile phase and the same solvent in the stationary liquid phase inside the pores of the filling (stationary phase). The range of pores determines the range of molecules for which separation can be achieved.
Molecules that are so small that they can penetrate all pores are eluted by total permeation volume (Vt). By contrast, molecules larger than the largest pores of filling migrate through a column only within the space between the particles of filling without being held in any way and are eluted by the elimination volume (V0 or dead volume). Molecular separation occurs between the elimination volume and the total permeation volume, usually within the first two thirds of this range.
Device. The base of the device is a chromatographic column of different lengths and internal diameter, thermostatic, filled, if necessary, with separation material capable of dividing in the appropriate range of molecular sizes, which is passed through the eluent at constant speed. One end of the column is usually connected to a suitable sample feed device such as a flow adapter, a dosing device with a sept or a dosing loop, and can also be connected to an appropriate pump for regulation of the elenite flow. Where appropriate, the sample may be sprayed directly to the surface of the column load free of liquid or, if the sample is denser than the eluent, may be placed under the eluent. The column output is usually connected to an appropriate detector associated with an automatic recorder that allows recording of relative concentrations of separated components of the sample. Detectors usually use photometric, refractometry or luminescence properties. If necessary, an automatic fractional pantograph may be connected.
The filling may be a soft carrier such as swelled gel, or a rigid carrier made up of glass, silica gel or a cross-linked organic polymer compatible with the solvent. Rigid carriers usually require pressurised systems to allow faster separation. The mobile phase is selected according to the type of sample of the separate environment and method of detection. Before performing the separation, the cartridge shall be adjusted and filled into the column as described in the article or in the manufacturer's instructions.
The criteria for assessing the suitability of the system are described in the state Chromatographic separation method (2.2.46). This status indicates the extent to which the parameters of the chromatographic system can be set to meet the requirements of the aptitude test.
Determination of relative representation of ingredients in mixtures
Separation shall be carried out according to the pharmacopoeia article. Where possible, the elution of the components shall be recorded continuously and the corresponding spades shall be measured. If the separation of the components of the sample is recorded through a physico-chemical characteristic to which all the monitored components contribute by equivalent responses (e.g. all components have the same specific absorbance), the relative quantity of each component shall be calculated by dividing the area of the pike of that component by the sum of the areas of the pikes of all the monitored components. If these responses differ for each component, the content shall be calculated using the calibration curves obtained with the calibration standards prescribed in the Pharmacopoeia article.
Determination of molecular weights
Elimination chromatography may be used to determine molecular weights by comparison with appropriate calibration standards prescribed in the Pharmacopoeia. The retention volumes of calibration standards are calculated according to the logarithms of the molecular weights of these standards. The graph usually approaches a line in the area between the elimination volume and the total permeation volume for the separation environment used. From the calibration curve, the molecular weight is determined. The molecular mass calibration curve applies only to a given macromolecules system a solvent substance that has been used under prescribed experimental conditions.
Determination of the distribution of polymer molecules
Elimination chromatography may be used to determine the distribution of polymer molecules. However, a comparison of the samples is only valid if the results are obtained under the same experimental conditions. The comparative substances used for calibration and methods for determining the distribution of molecular sizes are listed in the pharmacopoeia article.
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4. In Part 2 of the Annex, the test methods, Chapter 2.2. Physical and physico-chemical methods, the following Chapters 2.2.45 to 2.2.47 are inserted after Chapter 2.2.44:
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2.2.45 Supercritical Fluid Chromatography
Supercritical Fluid Chromatography (SFC) is a separation method where the mobile phase is a liquid in super critical or subcritical condition. The stationary phases in the column are either finely dispersed solid particles (such as silica gel or porous graphite), chemically modified stationary phases which are used in liquid chromatography or capillary columns chemically bound liquid stationary phases.
The SFC is based on an adsorption or distribution mechanism.
Device
The apparatus usually consists of a refrigerated pump system, a dosing device, chromatographic columns equipped with thermostat, detector, pressure regulator and data processing device (or integrator or recorder).
Pumps systems
The pump systems shall guarantee a constant flow rate of the mobile phase. In order to minimise pressure fluctuations, the system is equipped with a pulse damping device for the pressurised mobile phase. The hoses and all connections shall be able to withstand the pressure developed by the pumping system.
Microprocessor-controlled systems are capable of precisely delivering the mobile phase under either constant or changing conditions in accordance with the specified program. In the case of gradient elution, the pump system is used, which brings the solvent (s) from the respective containers. The mixing of solvents takes place either on the low pressure or high pressure pump side (s).
Dosing device
The injection is done directly at the beginning of the column with a dosing valve.
Stationary Phase
The stationary phases are in columns that have been described in the state of the liquid chromatography (2.2.29) (filling columns) and Gas chromatography (2.2.28) (capillary columns). Capillary columns have an internal diameter of not more than 100 μm.
Mobile Phase
Carbon dioxide is usually used, which sometimes contains polar modifiers such as methanol 2 propanol or acetonitrile. The composition, pressure (density), temperature and flow rate of the prescribed mobile phase are either constant throughout the chromatographic process (isocratic, izodensitic, isotermic elution) or may vary according to the defined programme (gradient elution of the modifier, pressure (density), temperature or flow rate).
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Regulation Information
| Citation | Decree of the Ministry of Health No. 180 / 2002 Coll., amending Decree of the Ministry of Health No. 1 / 1998 Coll., laying down requirements on quality, procedure for preparation, testing, storage and dosing of medicinal products (Czech Pharmacopoeia 1997), as amended |
|---|---|
| Regulation Type | Order |
| Author | - |
| Collection | Code of Laws |
| Date of Promulgation | 20.05.2002 |
|---|---|
| Effective from | 01.06.2002 |
| Effective until | - |
| Status | Valid |
Legal Areas:
Administrative law
Health
The regulation text is for informational purposes only.
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