How many chromatograms are needed to implement the HPLC technique. Coursework: High performance liquid chromatography of natural and waste water pollutants
"High performance liquid chromatography of natural and Wastewater»
Introduction
Chapter 1. Basic concepts and classification of liquid chromatography methods
1.1 Apparatus for liquid chromatography
Chapter 2. The essence of HPLC
2.1 Application
Chapter 3. Examples of using HPLC in the analysis of objects environment
Chapter 4 HPLC Instrumentation
Literature
Application
Introduction
Chromatographic Methods often indispensable for identification and quantification organic matter with a similar structure. The most widely used for routine analysis of environmental pollutants are gas and high performance liquid chromatography. Gas chromatographic analysis of organic pollutants in drinking and waste waters was initially based on the use of packed columns, later quartz capillary columns also became widespread. The internal diameter of capillary columns is usually 0.20-0.75 mm, length - 30-105 m. Optimum results in the analysis of contaminants in water are most often achieved when using capillary columns with different film thicknesses made of methylphenyl silicones with a content of phenyl groups of 5 and 50% . The sample injection system often becomes a vulnerable point in chromatographic techniques using capillary columns. Sample injection systems can be divided into two groups: universal and selective. The universal ones include injection systems with and without splitting the flow, “cold” injection into the column and evaporation with temperature programming. Selective injection uses purge with intermediate trapping, headspace analysis, etc. When using universal injection systems, the entire sample enters the column, with selective injection, only a certain fraction is introduced. The results obtained with selective injection are significantly more accurate, since the fraction that entered the column contains only volatile substances, and the technique can be fully automated.
Gas chromatographic detectors used in pollutant monitoring are often divided into universal detectors, which respond to each component in the mobile phase, and selective detectors, which react to the presence in the mobile phase of a certain group of substances with similar chemical characteristics. The universal ones include flame ionization, atomic emission, mass spectrometric detectors and infrared spectrometry. Selective detectors used in water analysis are electron-capture (selective to substances containing halogen atoms), thermionic (selective to nitrogen- and phosphorus-containing compounds), photoionization (selective to aromatic hydrocarbons), electrolytic conductivity detector (selective to compounds, containing halogen, sulfur and nitrogen atoms). The minimum detectable amounts of substances range from nanograms to picograms per second.
High Performance Liquid Chromatography(HPLC) is the ideal method for determining a large number thermally unstable compounds that cannot be analyzed using gas chromatography. Currently, modern agrochemicals, including methyl carbonates and organophosphorus insecticides, and other non-volatile substances, often become objects of analysis by liquid chromatography. High performance liquid chromatography (HPLC) is gaining ground among other methods used in environmental monitoring, also because it has bright prospects in terms of automating sample preparation.
CHAPTER 1. BASIC CONCEPTS AND CLASSIFICATION OF LIQUID CHROMATOGRAPHY METHODS
Liquid chromatography is divided into several classes depending on the type of stationary phase support. The simple instrumentation of paper and thin layer chromatography led to the widespread use of these methods in analytical practice. However, the great possibilities of column liquid chromatography stimulated the improvement of equipment for this classical method and led to the rapid introduction of HPLC. Passing the eluent through the column under high pressure made it possible to sharply increase the rate of analysis and significantly increase the separation efficiency due to the use of a finely dispersed sorbent. The HPLC method currently makes it possible to isolate, quantitatively and qualitatively analyze complex mixtures of organic compounds.
According to the mechanism of interaction of the separated substance (eluate) with the stationary phase, adsorption, distribution, ion-exchange, size-exclusion, ion-pair, ligand-exchange and affinity chromatography are distinguished.
Adsorption chromatography. Separation by adsorption chromatography is carried out as a result of the interaction of the substance to be separated with an adsorbent, such as aluminum oxide or silica gel, which have active polar centers on the surface. The solvent (eluent) is a non-polar liquid. The mechanism of sorption consists in a specific interaction between the polar surface of the sorbent and the polar (or capable of being polarized) regions of the molecules of the analyzed component (Fig. 1).
Rice. 1. Adsorption liquid chromatography.
Partition chromatography. In the distributive version of liquid chromatography, the separation of a mixture of substances is carried out due to the difference in their distribution coefficients between two immiscible phases - the eluent (mobile phase) and the phase located on the sorbent (stationary phase).
At normal-phase Partition liquid chromatography uses a non-polar eluent and polar groups grafted onto the surface of a sorbent (most often silica gel). Substituted alkylchlorosilanes containing polar groups, such as nitrile, amino group, etc., are used as silica gel surface modifiers (bonded phases) (Fig. 2). The use of bonded phases makes it possible to finely control the sorption properties of the surface of the stationary phase and achieve high separation efficiency.
Rice. 2. Partition chromatography with bonded phase (normal phase variant).
reversed phase liquid chromatography is based on the distribution of mixture components between the polar eluent and nonpolar groups (long alkyl chains) grafted onto the sorbent surface (Fig. 3).
Rice. 3. Partition chromatography with bonded phase (reversed-phase version).
Less widely used is a variant of supported phase liquid chromatography, in which a liquid stationary phase is applied to a stationary support.
Exclusive (gel penetrating) Chromatography is a variant of liquid chromatography in which the separation of substances occurs due to the distribution of molecules between the solvent located in the pores of the sorbent and the solvent flowing between its particles.
affine Chromatography is based on specific interactions of separated proteins (antibodies) with substances (antigens) grafted onto the surface of a sorbent (synthetic resin) that selectively form complexes (conjugates) with proteins.
Ion-exchange, ion-pair, ligand-exchange chromatography are used mainly in inorganic analysis.
Basic parameters of chromatographic separation.
The main parameters of chromatographic separation are the retention volume and the retention time of the mixture component (Fig. 4).
The retention time tR is the time elapsed from the moment the sample is injected into the column until the maximum of the corresponding peak is reached. Multiplying the retention time by the eluent volume velocity F, we get the retention volume VR:
The corrected retention time is the time elapsed from the moment the peak of the non-sorbable component appears to the peak of the corresponding compound:
tR" = tR - t0 ;
The normalized or corrected retention volume is the retention volume corrected for column dead volume V0, i.e. the retention volume of the non-sorbable component:
VR" = VR - V0;
The retention characteristic is also the capacitance factor k", defined as the ratio of the mass of the substance in the stationary phase to the mass of the substance in the mobile phase: k" = mn / mp;
The value of k" is easy to determine from the chromatogram:
The most important parameters of chromatographic separation are its efficiency and selectivity.
The efficiency of the column, measured by the height of the theoretical plates (HETP) and inversely proportional to their number (N), is higher, the narrower the peak of the substance emerging at the same retention time. The efficiency value can be calculated from the chromatogram using the following formula:
N = 5.54. (tR / 1/2) 2 ,
where tR- holding time,
w 1/2 - peak width at half height
Knowing the number of theoretical plates per column, the column length L, and the average sorbent grain diameter dc, it is easy to obtain the values of the theoretical plate equivalent height (HETP) and the reduced height (PETP):
HETP = L/N PHETP = HETP/d c
These characteristics make it possible to compare the efficiency of columns of different types, to evaluate the quality of the sorbent and the quality of filling the columns.
The selectivity of the separation of two substances is determined by the equation:
When considering the separation of a mixture of two components, the degree of separation RS is also an important parameter:
;
Peaks are considered resolved if the RS value is greater than or equal to 1.5.
The main chromatographic parameters relate the following equation for resolution:
;
The factors that determine separation selectivity are:
1) the chemical nature of the sorbent;
2) the composition of the solvent and its modifiers;
3) chemical structure and properties of the components of the mixture to be separated;
4) column temperature
1.1 Apparatus for liquid chromatography
In modern liquid chromatography, instruments of varying degrees of complexity are used - from the simplest systems to high-end chromatographs equipped with various additional devices.
On fig. Figure 4 shows a block diagram of a liquid chromatograph containing the minimum required set of components, in one form or another, present in any chromatographic system.
Rice. 4. Block diagram of a liquid chromatograph.
The pump (2) is designed to create a constant solvent flow. Its design is determined primarily by the operating pressure in the system. For operation in the range of 10-500 MPa, plunger (syringe) or piston type pumps are used. The disadvantage of the first is the need for periodic stops for filling with eluent, and the second is the great complexity of the design and, as a result, the high price. For simple systems with low operating pressures of 1-5 MPa, inexpensive peristaltic pumps are successfully used, but since it is difficult to achieve a constant pressure and flow rate, their use is limited to preparative tasks.
The injector (3) ensures that a sample of the mixture of separated components is injected into the column with a sufficiently high reproducibility. Simple "stop-flow" sample injection systems require the pump to be stopped and are therefore less convenient than Reodyne's loop pipettes.
The HPLC columns (4) are thick-walled stainless steel tubes capable of withstanding high pressure. An important role is played by the density and uniformity of packing the column with a sorbent. For low pressure liquid chromatography, thick-walled glass columns are successfully used. Temperature constancy is ensured by thermostat (5).
Detectors (6) for liquid chromatography have a flow cell in which some property of the flowing eluent is continuously measured. The most popular types of general purpose detectors are refractometers, which measure the refractive index, and spectrophotometric detectors, which measure the absorbance of a solvent at a fixed wavelength (usually in the ultraviolet region). The advantages of refractometers (and the disadvantages of spectrophotometers) include low sensitivity to the type of compound being determined, which may not contain chromophore groups. On the other hand, the use of refractometers is limited to isocratic systems (with a constant eluent composition), so the use of a solvent gradient is not possible in this case.
HPLC columns, which are most commonly used in environmental pollutant analysis, are 25 cm long and 4.6 mm inside diameter, and are filled with 5-10 µm spherical silica gel particles grafted with octadecyl groups. AT last years columns with a smaller inner diameter, filled with smaller particles, appeared. The use of such columns reduces the consumption of solvents and the duration of the analysis, increases the sensitivity and separation efficiency, and also facilitates the problem of connecting columns to spectral detectors. Columns with an internal diameter of 3.1 mm are equipped with a safety cartridge (precolumn) to increase the service life and improve the reproducibility of the analyses.
As detectors in modern HPLC instruments, a UV detector on a diode array, fluorescence, and electrochemical detectors are usually used.
It should be borne in mind that in practical work, separation often proceeds not by one, but by several mechanisms simultaneously. So, exclusion separation can be complicated by adsorption effects, adsorption - distribution, and vice versa. In this case, the greater the difference in the substances in the sample in terms of the degree of ionization, basicity or acidity, in terms of molecular weight, polarizability, and other parameters, the greater the likelihood of a different separation mechanism for such substances.
In practice, “reversed-phase” (partition) chromatography, in which the stationary phase is not polar, but the mobile phase is polar (i.e., the reverse of “straight-phase” chromatography), has become most widespread.
In most laboratories around the world, a group of 16 priority PAHs are analyzed by HPLC or CMS.
CHAPTER 2. ESSENCE OF HPLC
In high performance liquid chromatography (HPLC), the nature of the processes occurring in the chromatographic column is generally identical to the processes in gas chromatography. The difference is only in the use of a liquid as a stationary phase. Due to the high density of liquid mobile phases and the high resistance of the columns, gas and liquid chromatography differ greatly in instrumentation.
In HPLC, pure solvents or their mixtures are usually used as mobile phases.
To create a stream of pure solvent (or mixtures of solvents), called eluent in liquid chromatography, pumps are used, which are part of the chromatograph hydraulic system.
Adsorption chromatography is carried out as a result of the interaction of a substance with adsorbents, such as silica gel or aluminum oxide, which have active centers on the surface. The difference in the ability to interact with the adsorption centers of different sample molecules leads to their separation into zones in the process of moving with the mobile phase through the column. The division of the component zones achieved in this case depends on the interaction with both the solvent and the adsorbent.
Silica gel adsorbents with different volumes, surfaces, and pore diameters find the greatest application in HPLC. Aluminum oxide and other adsorbents are used much less frequently. The main reason for this:
Insufficient mechanical strength, which does not allow packaging and use at elevated pressures typical for HPLC;
silica gel compared to aluminum oxide has a wider range of porosity, surface and pore diameter; a significantly higher catalytic activity of aluminum oxide leads to a distortion of the analysis results due to the decomposition of the sample components or their irreversible chemisorption.
HPLC detectors
High performance liquid chromatography (HPLC) is used to detect polar non-volatile substances, which for some reason cannot be converted into a form convenient for gas chromatography, even in the form of derivatives. Such substances, in particular, include sulfonic acids, water-soluble dyes and some pesticides, such as phenyl-urea derivatives.
Detectors:
UV - diode array detector. The "matrix" of photodiodes (there are more than two hundred of them) constantly registers signals in the UV and visible region of the spectrum, thus ensuring the recording of UV-B spectra in the scanning mode. This makes it possible to continuously record, at high sensitivity, undistorted spectra of components rapidly passing through a special cell.
Compared to single-wavelength detection, which does not provide information about the "purity" of the peak, the ability to compare the full spectra of the diode array provides an identification result with a much greater degree of certainty.
Fluorescent detector. The great popularity of fluorescent detectors is due to the very high selectivity and sensitivity, and the fact that many environmental pollutants fluoresce (for example, polyaromatic hydrocarbons).
An electrochemical detector is used to detect substances that are easily oxidized or reduced: phenols, mercaptans, amines, aromatic nitro and halogen derivatives, aldehydes, ketones, benzidines.
Chromatographic separation of the mixture on the column due to the slow advance of the PF takes a long time. To speed up the process, chromatography is carried out under pressure. This method is called high performance liquid chromatography (HPLC).
Modernization of the equipment used in classical liquid column chromatography has made it one of the promising and modern methods of analysis. High performance liquid chromatography is a convenient method for separating, preparatively isolating, and performing qualitative and quantitative analysis of non-volatile thermolabile compounds of both low and high molecular weight.
Depending on the type of sorbent used in this method, 2 variants of chromatography are used: on a polar sorbent using a non-polar eluent (direct phase option) and on a non-polar sorbent using a polar eluent - the so-called reverse-phase high-performance liquid chromatography (RPHLC).
When the eluent passes to the eluent, the equilibrium under RPHLC conditions is established many times faster than under the conditions of polar sorbents and non-aqueous PFs. As a result of this, as well as the convenience of working with water and water-alcohol eluents, RPHLC has now gained great popularity. Most HPLC analyzes are carried out using this method.
Detectors. Registration of the output from the column of a separate component is performed using a detector. For registration, you can use the change in any analytical signal coming from the mobile phase and related to the nature and amount of the mixture component. Liquid chromatography uses such analytical signals as light absorption or light emission of the exiting solution (photometric and fluorimetric detectors), refractive index (refractometric detectors), potential and electrical conductivity (electrochemical detectors), etc.
The continuously detected signal is recorded by the recorder. The chromatogram is a sequence of detector signals recorded on the recorder tape, generated when individual components of the mixture exit the column. In the case of separation of the mixture, individual peaks are visible on the external chromatogram. The position of the peak on the chromatogram is used for the purpose of identification of the substance, the height or area of the peak - for the purpose of quantitative determination.
2.1 Application
HPLC finds the widest application in the following areas of chemical analysis (objects of analysis where HPLC has practically no competition are identified):
· Food quality control - tonic and flavor additives, aldehydes, ketones, vitamins, sugars, dyes, preservatives, hormones, antibiotics, triazine, carbamate and other pesticides, mycotoxins, nitrosoamines, polycyclic aromatic hydrocarbons, etc.
· Environmental protection - phenols, organic nitro compounds, mono- and polycyclic aromatic hydrocarbons, a number of pesticides, major anions and cations.
· Criminalistics - drugs, organic explosives and dyes, potent pharmaceuticals.
· Pharmaceutical industry - steroid hormones, practically all products of organic synthesis, antibiotics, polymer preparations, vitamins, protein preparations.
Medicine - the listed biochemical and medicinal substances and their metabolites in biological fluids (amino acids, purines and pyrimidines, steroid hormones, lipids) in the diagnosis of diseases, determining the rate of excretion of drugs from the body for the purpose of their individual dosage.
· Agriculture- determination of nitrate and phosphate in soils to determine the required amount of applied fertilizers, determination of the nutritional value of feed (amino acids and vitamins), analysis of pesticides in soil, water and agricultural products.
Biochemistry, bioorganic chemistry, genetic engineering, biotechnology - sugars, lipids, steroids, proteins, amino acids, nucleosides and their derivatives, vitamins, peptides, oligonucleotides, porphyrins, etc.
· Organic chemistry - all stable products of organic synthesis, dyes, thermolabile compounds, non-volatile compounds; inorganic chemistry (practically all soluble compounds in the form of ions and complex compounds).
· Quality control and safety of food products, alcoholic and non-alcoholic beverages, drinking water, household chemicals, perfumes at all stages of their production;
determination of the nature of pollution at the site of a man-made disaster or emergency;
detection and analysis of narcotic, potent, poisonous and explosives;
determination of the presence harmful substances(polycyclic and other aromatic hydrocarbons, phenols, pesticides, organic dyes, heavy, alkali and alkaline earth metal ions) in liquid effluents, air emissions and solid waste from enterprises and in living organisms;
· monitoring of processes of organic synthesis, oil and coal processing, biochemical and microbiological productions;
analysis of soil quality for fertilization, the presence of pesticides and herbicides in soil, water and products, as well as the nutritional value of feed; complex research analytical tasks; obtaining a micro amount of ultrapure substance.
CHAPTER 3. EXAMPLES OF USE OF HPLC IN THE ANALYSIS OF ENVIRONMENTAL OBJECTS
HPLC - a method for monitoring PAHs in environmental objects
For polycyclic aromatic hydrocarbons (PAHs), ecotoxicants of the 1st hazard class, extremely low levels of maximum permissible concentrations (MACs) have been established in natural objects. The determination of PAHs at the MPC level and below is one of the very complex analytical tasks and high-tech analysis methods (GC-MS, GC, HPLC) are used to solve them. When choosing a method for monitoring, in addition to the main characteristics under consideration - sensitivity and selectivity, expressness and economy are added, because. monitoring involves serial analysis. HPLC variant on short columns of small diameter in to a large extent meets the specified requirements. Using this method, the authors developed and certified methods for monitoring benzo[a]pyrene in three natural media: aerosol, snow cover, and surface waters. The methods are characterized by: simple unified sample preparation, including extraction of PAHs with organic solvents and concentration of the extract, direct introduction of a concentrated extract into a chromatographic column, the use of multiwavelength photometric detection in the UV region of the spectrum, identification of PAH peaks in chromatograms using two parameters, retention time and spectral ratio . The total error does not exceed 10% when determining benz[a]pyrene in aerosol in the concentration range from 0.3 to 450 ng/m up to 50 μg / m 2. For the case of simultaneous determination of priority PAHs (up to 12 compounds) and registration of inhomogeneous peaks of analytes, it was proposed to reseparate the extract with a change in the selectivity of the mobile phase, the detection wavelength, and the column temperature, taking into account the individual properties of the PAH being determined.
1 . Ambient air quality. Mass concentration of benzo[a]pyrene. The procedure for performing measurements by the HPLC method. Certificate of attestation MVI No. 01-2000.
2 . The quality of surface and treated wastewater. Mass concentration of benzo[a]pyrene. The procedure for performing measurements by the HPLC method. Certificate of attestation MVI No. 01-2001.
3 . Snow cover quality. Mass concentration of benzo[a]pyrene. The procedure for performing measurements by the HPLC method. Certificate of attestation MVI No. 02-2001.
Removal of Aniline from Aqueous Solutions Using Wastes of Aluminothermic Recovery of Rolled Copper Scale
The problem of removing hydrocarbons from wastewater is an urgent task. In many chemical, petrochemical and other industries, aniline and its derivatives are formed, which are toxic substances. Aniline is a highly toxic substance, MPC - 0.1 mg / m 3. Aniline and its derivatives are soluble in water and therefore cannot be removed by gravitational settling.
One of the best methods of wastewater treatment from organic pollutants is the use of inorganic and organic adsorbents capable of regeneration (aluminosilicates, modified clays, wood, fibers, etc.) and incapable of regeneration (activated carbon, macroporous polymeric materials, etc.). ).
Regenerated adsorbents can remove organic substances of different polarity from water. The search for effective adsorbents is an urgent task.
This report presents the results of a study in the field of using the milled copper scale of the Yerevan Cable Plant (OPMOERKZ) as aniline sorbents.
Chromatographic studies were carried out on an HPLC chromatograph / high performance liquid chromatography / systems (Waters 486 - detector, Waters 600S - controller, Waters 626 - Pump), on a 250 x 4 mm column filled with sorbents under study, mobile phase rate 1 ml/m / mobile phase are the solvents we are studying/, the detector is UV-254. UV spectroscopic analysis was carried out on a Specord-50 spectrophotometer, the spectra were obtained using the ASPECT PLUS computer program.
Precisely weighed portions of sorbents were added to certain volumes of aniline in water, the initial concentrations of which varied. The mixture was thoroughly shaken for 6 h. Then the sample was left to settle. Adsorption is completed in almost 48 hours. The amount of precipitated aniline was determined by UV spectrophotometric as well as refractive analysis.
At first, the adsorption properties of OPMOEPKZ were studied during the removal of aniline from a solution in carbon tetrachloride. It turned out that aniline absorbs sorbent 3 best of all (table).
Measurements were also carried out for aqueous solutions of aniline at concentrations of 0.01-0.0001 mol/l. The table shows data on a 0.01 M solution.
Absorption of aniline by various sorbents from 0.01 M aqueous solution of aniline at 20°C
Previously, it was found that adsorption within the indicated concentration ranges increases and depends linearly on the refractive index. The amount of aniline was determined from the refractive index versus molar concentration plot and corrected for both liquid chromatography and UV spectral analysis.
Sorbent 3 is the most active for aqueous solutions. The amount of adsorbed pollutant was calculated as the difference between the total amount of pollutant added to the initial solution and its residue in the final solution.
Methods for determining PAHs in environmental objects
Typically, gas chromatography (GC) and high performance liquid chromatography (HPLC) methods are used to determine PAHs. separation of the main 16 PAHs, sufficient for quantitative analysis, is achieved using either capillary columns in gas chromatography or high-performance columns used in HPLC. It must be remembered that a column that separates well the calibration mixtures of sixteen PAHs does not guarantee that they will also be well separated against the background of accompanying organic compounds in the samples under study.
In order to simplify the analysis, as well as to achieve High Quality obtained results, most analytical procedures contain the stage of preliminary isolation (separation) of PAHs from other groups of related compounds in samples. The most common methods used for this purpose are low-pressure liquid chromatography in liquid-solid or liquid-liquid systems using adsorption mechanisms, such as using silica gel or alumina, sometimes mixed mechanisms are used, such as adsorption and exclusion using Sephadex.
The use of pretreatment of samples makes it possible to avoid the influence of:
Completely non-polar compounds such as aliphatic hydrocarbons;
Moderately and strongly polar compounds, for example, phthalane, phenols, polyhydric alcohols, acids;
High molecular weight compounds such as, for example, resins.
Two types of detectors are mainly used in high performance liquid chromatography (HPLC): a fluorimetric detector or a photodiode bar spectrophotometric detector. The limit of detection of PAHs in fluorimetric detection is very low, which makes this method particularly suitable for the determination of trace amounts of polyaromatic compounds. However, classical fluorimetric detectors provide virtually no information about the structure of the compound under study. Modern designs make it possible to record fluorescence spectra that are characteristic of individual compounds, but they have not yet become widespread in the practice of routine measurements. A spectrophotometric detector with a photodiode line (PDL) makes it possible to record absorption spectra in the UV and visible spectral range, these spectra can be used for identification. Similar information can be obtained using fast scanning detectors.
When choosing an analytical technique for the separation, identification and quantification of these PAHs, the following conditions must be taken into account:
The level of determined contents in the studied samples;
The number of related substances;
Applied analytical procedure (measurement procedure);
Possibilities of the serial equipment.
Development of a Method for Determination of Alkaline Earth Elements and Magnesium by Ion High Performance Liquid Chromatography
The development and improvement of methods that allow solving problems of water analysis is an important problem in analytical chemistry. Development of high performance liquid chromatography high pressure stimulated the development of a new direction in ion-exchange chromatography, the so-called ion chromatography. The synthesis of sorbents for ion chromatography is difficult, since quite a lot of requirements are imposed on them. Due to the lack of commercially available high-performance cation exchangers, a dynamically modified reverse phase was used, for which a modifier was synthesized: N-hexadecyl-N-decanoyl-para-amino-benoylsulfonic acid ethyl-diisopropylammonium (DGDASC), where the hydrophobic amine containing the SO 3 - group, capable of cation exchange. After passing the modifier solution, the absorption at l = 260 nm reached 6.4 units of optical density (° E) reaching a plateau. The calculated ion exchange capacity is 15.65 µmol. Since the cations of alkaline earth elements and magnesium do not absorb in the UV region of the spectrum, indirect UV detection was used using the synthesized UV absorbing eluent 1,4-dipyridinium butane bromide (DPB bromide). Since halogen ions destroy the steel parts of the column, the bromide ion of 1,4-dipyridinium butane was replaced by an acetate ion. When the column is washed with eluent, the counterion of the modifier, ethyldiisopropylammonium, is replaced by the UV-absorbing ion 1,4-dipyridiniumbutane. Separation of cations was carried out at the optimal wavelength l = 260 nm on a scale of 0.4 A in the “scale folding” mode; the polarity of the recorder was reversed. The separation of all the studied cations was achieved with the introduction of a complexing additive - oxalic acid. The detection limits of Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ are 8 µg/l; 16 µg/l; 34 µg/l; 72 µg/l, respectively. Under the selected conditions, tap water was analyzed, the content of Ca 2+ in which is 10.6 +1.9 mg-ion/l, Mg 2+ -2.5 + mg-ion/l. The reproducibility error does not exceed -2.2% for Ca 2+ and 1.4% for Mg 2+.
Analysis of cadmium complexes in the environment
To study the mechanisms of migration of heavy metals in the biosphere, data on the chemical forms of the existence of metals in nature are needed. Difficulties in the analysis of compounds of one of the most toxic metals- cadmium - due to the fact that it forms unstable complexes, and when you try to isolate them, natural balances are distorted. In this work, cadmium compounds in soil and plants were studied using a technique based on the chromatographic separation of extracts followed by the identification of components by chemical analysis. This approach made it possible not only to identify the chemical forms of cadmium, but also to trace their transformations in environmental objects.
OH-groups of carbohydrates and polyphenols (including flavonoids), C=O, phosphates, NH 2 , NO 2 , SH-groups are coordinated with cadmium in biosphere objects. For the purposes of this study, a set of model ligands representing these classes of compounds was compiled. The interaction of model ligands with water-soluble cadmium salts was studied by UV spectroscopy and HPLC.
To isolate cadmium compounds, extraction with specially selected (not forming complexes with Cd) solvents was used. In this way, cadmium can be separated from all heavy metals, except for its close chemical analogue, zinc. Cadmium- and zinc-containing peaks in the chromatograms of the obtained extracts were detected by binding metals in the form of their dithizonates. To separate from zinc, the difference in the stability of the Cd and Zn complexes at pH 6–8 was used. The isolated Cd compounds were identified by HPLC with pH changes during elution. The analysis of cadmium compounds with the components of soils and plant tissues was performed, and the substances produced by plants in response to an increase in the intake of cadmium from the soil were identified. It has been shown that flavonoids, in particular tricine, are protective agents in cereals, alkoxy derivatives of cysteine in legumes, and both polyphenols and thiols in cruciferous plants.
CHAPTER 4. HPLC EQUIPMENT
SERIES ACCELA
The new ACCELA Ultra High Performance Liquid Chromatograph is capable of operating over a wide range of flow rates and pressures, providing both typical HPLC separations on conventional columns and ultra-fast and efficient separations on columns with a sorbent particle size of less than 2 µm at ultra-high pressures (greater than 1000 atm.).
The system includes a quaternary gradient inert pump capable of delivering pressures in excess of 1000 atm and with a delay volume of only 65 µl, providing high speed chromatographic separations. Autosampler ACCELA capable of operating in a sample injection cycle of 30 seconds and provides the highest injection reproducibility. Diode Array Detector Accela PDA with a minimized flow cell volume (2 µl) is optimized for high-speed chromatography, uses patented LightPipe technology and maintains the symmetrical peak shape that comes with a flawless chromatography system and columns.
The system pairs perfectly with mass spectrometers to create the most powerful and best LC/MS systems available in the world.
1.9 µm UHP columns available from Thermo Electron for all applications
SERIES TSP
The modular principle of construction of HPLC instruments allows the customer to flexibly complete equipment for solving any analytical problems, and when they change, it can be quickly and economically modified. The wide range of modules includes pumps from isocratic to quaternary gradient, from micro-column to semi-preparative, all available detectors, sample injection systems from manual injectors to autosamplers with the ability to handle any sample, powerful software for processing measurement results and managing all modules of the system. All modules are certified according to CSA, TUF/GS, FCC(EMI), VDE (EMI), ISO-9000, they are compact, have a modern design, are easy to operate, equipped with a built-in display and self-diagnostic system, allow you to create and store task methods in memory parameters. They meet the criteria of "Exemplary Laboratory Practice" (GLP) and are listed in the Register of Measuring Instruments of the Russian Federation. Measurement protocols are issued in accordance with the Pharmacopoeias of England, USA, Germany and France.
TSP modular systems are characterized by the highest reliability and stability in operation.
The combination of modules provides the analyst with all the advantages of an integrated system on the one hand and the flexibility of a modular system on the other. Whichever field of application of High Performance Liquid Chromatography (HPLC) - pharmacology, biotechnology, environmental analysis, clinical analysis, food and beverage analysis, petrochemical and chemical analysis - this instrument is used, it is always optimally configured in order to meet the highest requirements.
Both research and high performance routine systems provide:
Highly efficient solvent degassing
Ability to work with small and ultra-small sample quantities
Highest sensitivity, both with UV/VIS detector and diode array (with the famous LightPipe technology with a choice of 1 or 5 cm optical path length)
Working with different columns
Highest Quantitative Accuracy
Possibility of automatic work with different sample volumes
RMS retention time error less than 0.3%
Minimum working area occupied by the system
Highest reliability and parameter stability.
Surveyor LC Pump- An HPLC pump with the best retention time reproducibility of any 4-component gradient pump available in the world. An integrated quad-channel vacuum degasser and pulsation damper provide excellent baseline stability for maximum quantitation sensitivity and accuracy.
The autosampler provides the highest performance and analysis flexibility. A wide range of sample trays, from standard vials to 96- and 384-well microplates, covers the needs of virtually all applications. New technology provides almost no loss of sample injection, almost 5 µl of sample is injected with an autosampler from a total sample volume of 5 µl.
SURVEYOR
UV/Visual Detector and PDA (Diode Array Detector)
Surveyor UV/Vis- variable wavelength ultraviolet and visible light detector is a combination of economy and reliability with the highest sensitivity of LightPipe technology. A wide selection of flow cells makes this detector versatile for all applications from those using capillary or microcolumn chromatography to semi-preparative and preparative.
Surveyor PDA The detector is the most sensitive among all HPLC diode array detectors. Dual-lamp source optics seamlessly cover the entire wavelength range from 190 to 800 nm. The fiber optic beamformer provides excellent optical resolution without sacrificing sensitivity.
Surveyor RI refractometric detector with a minimum volume thermostated cuvette with full electronic control from a computer.
Surveyor FL fluorometric scanning detector with the highest sensitivity and detection capability for fluorescence, chemiluminescence and phosphorescence.
A wide range of autosamplers allows you to work with both conventional vials and 96-position plates, widely used in biochemistry and clinical practice. Handling is facilitated by the use of similar SPE sample preparation plates.
400 Electric drive, Valco loop (20 µl - standard) with the possibility of partial filling.
Carousel 96 samples.
Electric drive, column thermostat, Valco loop (100 µl - standard) with the possibility of partial filling. AutoMix mode for sample preparation. Sample carousel: 84 x 2 ml (samples) + 3 x 10 ml (reagents). Built-in column thermostat. 420
Loop autosampler for research work with the ability to work in the modes of full, partial filling and introduction of microliter samples. A wide range of carousels (standard - 96 samples).
Tablet autosampler for 96- and 384-position plates. Injection of the sample into the pressure loop, the possibility of introducing samples of less than 1 µl. Possibility to install a tablet feeder. HPLC
Major manufacturers of HPLC equipment
· Waters - high performance chromatography, mass spectrometry, columns, solid phase extraction;
Varian, Inc. - chromatographs and columns, accessories for solid phase extraction;
· Agilent Technologies - chromatographs and columns;
· Hypersil - columns and sorbents.
· Merck KGaA - TLC plates and accessories for TLC, columns, sorbents mobile phases for HPLC, accessories for solid phase extraction
· Dionex - equipment and columns for HPLC, especially for ion chromatography.
Literature
1.Pilipenko A.T., Pyatnitsky I.V. Analytical chemistry. In two books: kn..1 - M .: Chemistry, 1990, -480s.
1. Pilipenko A.T., Pyatnitsky I.V. Analytical chemistry. In two books: kn..2 - M .: Chemistry, 1990, -480s.
2. Vasiliev V.P. Analytical chemistry. At 2 pm Part 2. Physical and chemical methods of analysis: Proc. for Khimko - technol. specialist. universities. - M .: Higher. school, 1989. - 384p.
3. Hydrochemical materials. Volume 100. Methods and technical means of operational monitoring of surface water quality. L.: Gidrometeo-izdat, 1991. - 200p.
4. Lurie Yu.Yu. Analytical chemistry of industrial wastewater / Yu.Yu. Lurie; M.: Chemistry Yu, 1984. - 448s.
5. Ewing G. Instrumental methods of chemical analysis / Per. from English. M.: Mir, 1989. - 348 p.
6. Gorelik D.O., Konopelko L.A., Pankov E.D. Environmental monitoring. In 2 vols. St. Petersburg: Christmas. 2000. - 260 p.
7. Aivazov B.V. Introduction to chromatography. M.: Higher. school, 1983. - 450 p.
8. Goldberg K.A., Vigdergauz M.S. Introduction to gas chromatography. M.: Chemistry, 1990. - 329 p.
9. Stolyarov B.V. and others // Practical gas and liquid chromatography. St. Petersburg: St. Petersburg State University, 1998. - S. 81.
11. Gorshkov A.G., Marinaite I.I. HPLC - a method for monitoring PAHs in environmental objects
12. Torosyan G. O., Martirosyan V. A., Aleksanyan A. R., Zakaryan M. O. Removal of aniline from aqueous solutions using waste products of aluminothermal reduction of rolling copper scale
13. L.A. Turkina, G.N. Koroleva Development of a method for the determination of alkaline earth elements and magnesium by ion high performance liquid chromatography
14. Dultseva G.G., Dubtsova Yu.Yu., Skubnevskaya G.I. Analysis of cadmium complexes in the environment
Application
DETERMINATION OF CLOMAZONE IN WATER BY CHROMATOGRAPHIC METHODS
METHODOLOGICAL INSTRUCTIONS MUK 4.1.1415-03
1. Prepared by: Federal Scientific Center for Hygiene. F.F.
Erisman; Moscow Agricultural Academy. K.A.
Timiryazev; with the participation of the Department of State Sanitary and Epidemiological Surveillance of the Ministry of Health of Russia. The developers of the methodology are listed at the end.
3. Approved by the Chief State Sanitary Doctor
Russian Federation, First Deputy Minister of Health of the Russian Federation, acad. RAMS G.G. Onishchenko June 24, 2003
5. Introduced for the first time.
1. Introduction
Manufacturer: FMS (USA).
Trade name: COMMAND.
Active ingredient: clomazone.
2-(2-chlorobenzyl)-4,4-dimethyl-3-isoxalidin-3-one(IUPAC)
Light brown viscous liquid.
Melting point: 25 -C.
Boiling point: 275 -C.
Vapor pressure at 25 -C: 19.2 MPa.
Partition coefficient n-octanol/water: K logP = 2.5.
Highly soluble in acetone, hexane, ethanol, methanol,
chloroform, dichloromethane and acetonitrile; solubility in water -
1.10 g/cu. dm. Stable at room temperature for at least 2 years, at 50 -C - at least 3 months.
Brief toxicological profile: Acute oral
toxicity (LD) for rats - 1369 - 2077 mg/kg; acute dermal
toxicity (LD) for rats - more than 2000 mg/kg; acute
inhalation toxicity (LC) for rats - 4.8 mg / cu. dm (4 hours).
Hygienic standards. MPC in water - 0.02 mg / cu. dm.
Scope of the drug. Clomazone is a selective herbicide used to control cereals and dicotyledonous weeds in soybean and rice crops during pre-emergence or pre-sowing application.
2. Method for determining clomazone in water
chromatographic methods
2.1. Key points
2.1.1. The principle of the technique
The technique is based on the extraction of clomazone from the analyzed sample with hexane, concentration of the extract and subsequent quantitative determination by alternative methods:
high performance liquid chromatography (HPLC) with
ultraviolet detector, gas liquid chromatography (GLC) with a constant recombination rate detector or thin layer chromatography (TLC). Quantitative determination is carried out by the method of absolute calibration.
2.1.2. Method selectivity
Under the proposed conditions, the method is specific in the presence of global environmental pollutants: chlorine derivatives of cycloparaffins (HCH isomers), diphenyl compounds (DDT and its derivatives), their metabolites - polychlorinated benzenes and phenols, as well as in the presence of sodium trichloroacetate, which can be used on crops in as a herbicide.
2.1.3. Metrological characteristic of the method (P = 0.95)
Reagents, solutions and materials
Clomazone with the content of d. 99.8%
(FMS, USA)
Nitrogen, och GOST 9293-79
Water ammonia, 25%, h GOST 1277-81
Acetone, h GOST 2603-79
n-Hexane, h GOST 2603-79
Hydrogen peroxide, 30% aqueous solution GOST 10929-77
Isopropyl alcohol, chemically pure TU 6-09-402-75
Sulfuric acid, chemically pure GOST 4203-77
Hydrochloric acid (hydrochloric), chemically pure GOST 3118-77
Methyl alcohol, chemically pure GOST
Sodium hydroxide, chemically pure, 25% aqueous solution GOST 4323-77
Sodium sulfate anhydrous, chemically pure GOST 1277-81
Silver nitrate, chemically pure GOST 1277-81
2-Phenoxymethanol, h TU 6-09-3688-76
Chromaton N-AW-DMCS (0.16 - 0.20 mm)
with 5% SE-30, Hemapol, Czech Republic
Chromaton N-AW-DMCS (0.16 - 0.20 mm) with 1.5
OV-17 + 1.95% QF-1, Hemapol, Czech Republic
Plates for HPTLC (USSR)
Records "Kieselgel 60 F-254" (Germany)
Records "Silufol" Czech Republic
Paper filters "white tape", ashless and pre-washed with hexane TU 6-09-2678-77
2.3. Cutlery, equipment, utensils
Liquid chromatograph Milichrome
with UV detector
Chromatographic steel column,
length 64 mm, inner diameter 2 mm,
filled with Silasorb 600, grain size 5 µm
Gas chromatograph series "Color" or
similar, equipped with a constant detector
recombination rate (RPR) with a limit
detection by lindane 4 x 10 g/cu. cm
Chromatographic glass column, length
1 or 2 m, inner diameter 2 - 3 mm
Microsyringe type MSH-10, capacity 10 µl TU 5E2-833-024
Apparatus for shaking type AVU-6s TU 64-1-2851-78
Water bath TU 64-1-2850-76
Analytical balance type VLA-200 GOST 34104-80E
Chromatographic chamber GOST 10565-74
Water jet pump GOST 10696-75
Mercury-quartz irradiator type OKN-11 TU 64-1-1618-77
Spray guns glass GOST 10391-74
Rotary vacuum evaporator IR-1M
or similar TU 25-11-917-76
Compressor unit TU 64-1-2985-78
Drying cabinet TU 64-1-1411-76E
Dividing funnels GOST 3613-75
Volumetric flasks, with a capacity of 100 ml GOST 1770-74
Measuring cylinders, with a capacity of 10, 50 ml GOST 1770-74E
Pear-shaped flasks with a thin section,
with a capacity of 100 ml GOST 10394-72
Flasks conical, with a capacity of 100 ml GOST 22524-77
Centrifuge test tubes, measured GOST 25336-82E
Pipettes, with a capacity of 0.1, 1, 2, 5 and 10 ml GOST 20292-74
Chemical funnels, conical, diameter
34 - 40 mm GOST 25336-82E
2.4. Sample selection
Sampling, storage and preparation of samples are carried out in accordance with
"Unified rules for sampling agricultural products, food products and environmental objects for the determination of trace amounts of pesticides", approved for N 2051-79 of 21.08.79
Samples taken can be stored in the refrigerator for up to 5 days. Before analysis, water (in the presence of suspension) is filtered through a loose paper filter.
2.5. Preparation for definition
2.5.1. HPLC method
2.5.1.1. Mobile phase preparation for HPLC
In a volumetric flask with a capacity of 100 ml, 5 ml of isopopanol and 5 ml of methanol are placed with a pipette, topped up to the mark with hexane, mixed, filtered.
2.5.1.2. Column conditioning
Rinse the HPLC column with hexane-methanol-isopropanol (90:5:5, v/v) for 30 min. at a solvent feed rate of 100 µl/min.
2.5.2. GLC method. Column preparation and conditioning
The finished packing (5% SE-30 on Chromaton N-AW-DMCS) is poured into a glass column, compacted under vacuum, the column is installed in the chromatograph thermostat, not connected to the detector, and stabilized in a stream of nitrogen at a temperature of 250 -C for 10 - 12 noon
2.5.3. TLC method
2.5.3.1. Preparation of developing reagents
2.5.3.1.1. Developing reagent No. 1
1 g of silver nitrate is dissolved in 1 ml of distilled water, 10 ml of 2-phenoxymethanol, 190 ml of acetone, 1-2 drops of hydrogen peroxide are added, the solution is stirred and transferred into a dark glass bottle.
2.5.3.2.2. Developing reagent N 2
0.5 g of silver nitrate is dissolved in 5 ml of distilled water in a 100 ml volumetric flask, 10 ml of 25% aqueous ammonia is added, the solution is adjusted to 100 ml with acetone, mixed and transferred to a dark glass bottle.
2.5.3.2. Mobile phase preparation for TLC
In a volumetric flask with a capacity of 100 ml add 20 ml of acetone and add hexane to the mark, mix. The mixture is poured into the chromatographic chamber with a layer of no more than 6 - 8 mm in 30 minutes. Before starting chromatography.
2.5.4. Preparation of standard solutions
A 100 µg/mL clomazone stock standard solution is prepared by dissolving 0.010 g of a preparation containing 99.8% AI in hexane in a 100 mL volumetric flask. The solution is stored in the refrigerator for a month.
Working standard solutions with a concentration of 0.4; 1.0; 2.0; 4.0; 10.0; 20 and 40.0 µg/ml are prepared from clomazone stock standard solution by appropriate serial dilutions with hexane.
Working solutions are stored in the refrigerator for no more than a month.
2.5.5. Construction of a calibration graph
2.5.5.1. Calibration curve A (measurement according to paragraph 2.7.1, HPLC)
To build a calibration graph, 5 µl of a working standard solution of clomazone with a concentration of 4.0 is injected into the chromatograph injector; 10.0; 20.0 and 40 µg/ml.
2.5.5.2. Calibration curve B (measurement according to paragraph 2.7.2, GLC)
To build a calibration graph, 5 µl of a working standard solution of clomazone with a concentration of 0.4 is injected into the chromatograph evaporator; 1.0; 2.0; 4.0 and 10.0.
Carry out at least 5 parallel measurements. Find the average height of the chromatographic peak for each concentration. Build a calibration graph (A or B) of the dependence of the height of the chromatographic peak in mm on the concentration of clomazone in solution in µg/ml.
2.6. Definition Description
100 ml of the analyzed water sample is placed in a separating funnel with a capacity of 250 ml, 10 ml of a 25% aqueous solution of sodium hydroxide is added, mixed and 20 ml of n-hexane are added. The funnel is shaken for 3 minutes, after phase separation, the hexane layer is poured into a pear-shaped flask with a capacity of 100 ml, passing it through a layer of anhydrous sodium sulfate placed in a conical funnel on a pleated filter paper. The extraction of the drug from the aqueous sample is repeated twice more using 20 ml of n-hexane. The combined hexane extract is evaporated on a rotary vacuum evaporator at a temperature of 40 -C almost to dryness, the residue is blown off with a stream of air or nitrogen of special purity. The dry residue is dissolved in 0.1 (HPLC, TLC) or 0.25 ml (GLC) n-hexane and analyzed by one of the chromatographic methods.
2.7. Chromatography conditions
Liquid chromatograph with ultraviolet detector Milichrom (Russia).
Steel column 64 mm long, inner diameter 2 mm,
filled with Silasorb 600, grain size 5 microns.
Column temperature: room.
Mobile phase: hexane-isopropanol-methanol (90:5:5, v/v).
Eluent flow rate: 100 µl/min.
Operating wavelength: 240 nm.
Sensitivity: 0.4 units absorption on the scale.
Injection volume: 5 µl.
Clomazone exit time: about 6 min.
Linear detection range: 20 - 200 ng.
Samples producing peaks greater than the 40 µg/mL standard solution are diluted with HPLC mobile phase.
Gas chromatograph "Tsvet-570" with detector of constant ion recombination rate.
Glass column 1 m long, 3 mm inner diameter, filled with Chromaton N-AW-DMCS with 5% SE-30 (0.16 - 0.20 mm).
The working scale of the electrometer is 64 x 10 10 Ohm.
Recorder tape speed 200 mm/h.
Column thermostat temperature - 190 -С
detector - 300 -C
evaporator - 220 -C
The speed of the carrier gas (nitrogen) - 60 ml / min.
The volume of the injected sample is 5 µl.
The exit time of clomazone is 2.5 minutes.
Linear detection range: 2 - 50 ng.
Samples that produce peaks greater than the 10 µg/mL standard solution are diluted with hexane.
To improve the accuracy of clomazone identification in the presence of gamma-HCCH having a close retention time in the sample, clomazone is removed from the sample by treatment with concentrated sulfuric acid. Re-analysis of the sample allows you to establish the contribution of clomazone to the primary chromatographic signal.
Hexane solution in a flask, obtained according to paragraph 2.6 quantitatively
(or an aliquot thereof) is applied to chromatographic plates "Silufol", "Kieselgel 60F-254" or "Plates for HPTLC". Nearby, standard solutions are applied in a volume corresponding to the content of clomazone 1, 2, 5 and 10 μg. The plate is placed in a chromatographic chamber containing a mixture of n-hexane-acetone (4:1, v/v). After the development of the chromatogram, the plate is removed from the chamber, placed under draft until the solvents evaporate, then treated with one of the developing reagents and placed under an ultraviolet lamp for 5 minutes. The localization zone of the drug on the plates "Silufol", "Plates for HPTLC" and "Kieselgel 60F-254" appears as gray-brown spots with Rf values of 0.35, 0.85 and 0.43, respectively. To determine clomazone by TLC, you can use plates "Alugram" and "Polygram" (manufactured by Germany). The Rf value of clomazone on these plates is 0.37 and 0.38, respectively.
3. Safety requirements
It is necessary to follow generally accepted safety rules when working with organic solvents, toxic substances, electric heaters.
4. Measurement error control
Operational control of the error and reproducibility of measurements is carried out in accordance with the recommendations of MI 2335-95. GSI "Internal quality control of the results of quantitative chemical analysis".
5. Developers
Yudina T.V., Fedorova N.E. (FNTSG named after F.F. Erisman).
Davidyuk E.I. (UkrNIIGINTOX, Kyiv); Kisenko M.A., Demchenko V.F. (Institute of Occupational Medicine of the Academy of Sciences and the Academy of Medical Sciences of Ukraine, Kyiv).
Liquid adsorption chromatography on a column
The separation of a mixture of substances in an adsorption column occurs as a result of their difference in sorbability on a given adsorbent (in accordance with the law of adsorption substitution established by M. S. Tsvet).
Adsorbents are porous bodies with a highly developed inner surface that retain liquids through intermolecular and surface phenomena. These can be polar and non-polar inorganic and organic compounds. Polar adsorbents include silica gel (dried gelatinous silicon dioxide), aluminum oxide, calcium carbonate, cellulose, starch, etc. Non-polar sorbents - activated carbon, rubber powder and many others obtained synthetically.
The adsorbents are subject to the following requirements: S they must not enter into chemical reactions with the mobile phase and the substances to be separated; S must have mechanical strength; S grains of the adsorbent must be of the same degree of dispersion.
When choosing the conditions for the chromatographic process, the properties of the adsorbent and adsorbed substances are taken into account.
In the classical version of liquid column chromatography (LCC), an eluent (PF) is passed through a chromatographic column, which is a glass tube 0.5–5 cm in diameter and 20–100 cm long, filled with a sorbent (NP). The eluent moves under the influence of gravity. The speed of its movement can be adjusted by the crane at the bottom of the column. The mixture to be analyzed is placed at the top of the column. As the sample moves through the column, the components separate. At certain intervals, fractions of the eluent released from the column are taken, which are analyzed by any method that allows measuring the concentrations of analytes.
Column adsorption chromatography is currently used mainly not as an independent method of analysis, but as a method of preliminary (sometimes final) separation of complex mixtures into simpler ones, i.e. to prepare for analysis by other methods (including chromatographic). For example, a mixture of tocopherols is separated on an alumina column, the eluent is passed, and the α-tocopherol fraction is collected for subsequent photometric determination.
Chromatographic separation of the mixture on the column due to the slow advance of the PF takes a long time. To speed up the process, chromatography is carried out under pressure. This method is called High Performance Liquid Chromatography (HPLC)
The modernization of the equipment used in classical liquid column chromatography has made it one of the most promising and modern methods analysis. High performance liquid chromatography is a convenient method for the separation, preparative isolation, and qualitative and quantitative analysis of non-volatile, thermolabile compounds of both low and high molecular weight.
Depending on the type of sorbent used, this method uses 2 chromatography options: on a polar sorbent using a non-polar eluent (direct phase option) and on a non-polar sorbent using a polar eluent - the so-called reversed-phase high-performance liquid chromatography (RP HPLC).
When the eluent passes to the eluent, the equilibrium under RPHLC conditions is established many times faster than under the conditions of polar sorbents and non-aqueous PFs. As a result of this, as well as the convenience of working with water and water-alcohol eluents, RPHLC has now gained great popularity. Most HPLC analyzes are carried out using this method.
Instrumentation for HPLC
A set of modern equipment for HPLC, as a rule, consists of two pumps 3,4 (Fig. 7.1.1.1), controlled by a microprocessor 5, and supplying eluent according to a specific program. Pumps create pressure up to 40 MPa. The sample is injected through a special device (injector) 7 directly into the eluent flow. After passing through the chromatographic column 8, the substances are detected by a highly sensitive flow detector 9, the signal of which is recorded and processed by the microcomputer 11. If necessary, fractions are automatically selected at the time of the peak output.
Columns for HPLC are made of stainless steel with an inner diameter of 2 - 6 mm and a length of 10-25 cm. The columns are filled with a sorbent (NF). Silica gel, alumina, or modified sorbents are used as NF. Silica gel is usually modified by chemically introducing various functional groups into its surface.
Detectors. Registration of the output from the column of a separate component is performed using a detector. For registration, you can use the change in any analytical signal coming from the mobile phase and related to the nature and amount of the mixture component. Liquid chromatography uses such analytical signals as light absorption or light emission of the exiting solution (photometric and fluorimetric detectors), refractive index (refractometric detectors), potential and electrical conductivity (electrochemical detectors), etc.
The continuously detected signal is recorded by the recorder. A chromatogram is a sequence of detector signals recorded on a tape recorder, which are generated when individual components of a mixture leave the column. In the case of separation of the mixture, separate peaks are visible on the external chromatogram. The position of the peak on the chromatogram is used for the purposes of identifying the substance, the height or area of the peak is used for the purposes of quantitative determination.
Qualitative Analysis
The most important characteristics of the chromatogram - the retention time tR and the retention volume associated with it - reflect the nature of the substances, their ability to sorption on the material of the stationary phase and, therefore, under constant chromatography conditions, they are a means of identifying the substance. For a given column with a certain flow rate and temperature, the retention time of each compound is constant (Figure 7.1.1.2), where t.R(A) is the retention time of component A of the analyzed mixture from the moment it is injected into the column until the maximum peak appears at the column outlet, 1K( ss) - retention time of the internal standard (substance initially absent in the analyzed mixture), h - peak height (mm), ab - peak width at half its height, mm.
To identify a substance by chromatogram, standard samples or pure substances are usually used. Compare the retention time of the unknown IR* component with the IRCT retention time of the known substances. But more reliable identification by measuring the relative retention time
In this case, a known substance (internal standard) is first introduced into the column and its retention time tR(Bc) is measured, then the test mixture is chromatographically separated (chromatographed), to which the internal standard is preliminarily added. The relative retention time is determined by formula (7.1.1.1).
Quantitative Analysis
This analysis is based on the dependence of the peak height h or its area S on the amount of substance. For narrow peaks, measurement h is preferable, for broad blurry peaks - S. The peak area is measured in different ways: by multiplying the peak height (h) by its width (ai / 2), measured at half its height (Fig. 7.2.3); planning; using an integrator. Modern chromatographs are equipped with electrical or electronic integrators.
Three methods are mainly used to determine the content of substances in a sample: the absolute calibration method, the internal normalization method, and the internal standard method.
The absolute calibration method is based on a preliminary determination of the relationship between the amount of the introduced substance and the area or height of the peak on the chromatogram. A known amount of the calibration mixture is introduced into the chromatogram and the areas or heights of the obtained peaks are determined. Build a graph of the area or height of the peak from the amount of injected substance. The test sample is analyzed, the area or height of the peak of the component to be determined is measured, and its amount is calculated based on the calibration curve.
This method provides information only on the relative content of the component in the mixture, but does not allow determining its absolute value.
The internal standard method is based on the comparison of a selected peak parameter of an analyte with the same parameter of a standard substance introduced into the sample in a known amount. A known amount of such a standard substance is introduced into the test sample, the peak of which is sufficiently well separated from the peaks of the components of the test mixture
The last two methods require the introduction of correction factors characterizing the sensitivity of the detectors used to the analyzed substances. For different types of detectors and different substances, the sensitivity coefficient is determined experimentally.
Liquid adsorption chromatography also uses the analysis of fractions of solutions collected at the moment the substance exits the column. The analysis can be carried out by various physicochemical methods.
Liquid adsorption chromatography is used primarily for the separation of organic substances. This method is very successful in studying the composition of oil, hydrocarbons, effectively separating trans- and cis-isomers, alkaloids, etc. HPLC can be used to determine dyes, organic acids, amino acids, sugars, pesticide and herbicide impurities, medicinal substances and other contaminants in food products.
(OFS 42-0096-09)
High performance liquid chromatography (HPLC) is a column chromatography method in which the mobile phase (MP) is liquid
bone moving through a chromatographic column filled with unsupported
the visual phase (sorbent). HPLC columns are characterized by high hydraulic pressure at the column inlet, therefore HPLC is sometimes called
called "High Pressure Liquid Chromatography".
Depending on the mechanism of separation of substances, the following are distinguished:
general HPLC options: adsorption, distribution, ion-exchange,
exclusive, chiral, etc.
In adsorption chromatography, the separation of substances occurs due to their different ability to be adsorbed and desorbed with increasing
surface of the adsorbent with a developed surface, for example, silica gel.
In partition HPLC, separation occurs due to the difference in the distribution coefficients of the substances to be separated between the immobile
(usually chemically grafted onto the surface of a fixed carrier) and
mobile phases.
By polarity, PF and NF HPLC are divided into normal-phase and ob-
phase-rotation.
Normal-phase is called a variant of chromatography, in which
use a polar sorbent (for example, silica gel or silica gel with added
twisted NH2 - or CN-groups) and non-polar PF (for example, hexane with different
personal supplements). In the reversed-phase variant of chromatography,
use non-polar chemically modified sorbents (for example,
non-polar alkyl radical C18 ) and polar mobile phases (for example,
methanol, acetonitrile).
In ion-exchange chromatography, the molecules of the substances of the mixture, dissociation
in solution into cations and anions, are separated when moving through
sorbent (cation exchanger or anion exchanger) due to their different exchange rates with ionic
mi groups of the sorbent.
In exclusion (sieve, gel-penetrating, gel-filtration)
Chromatography molecules of substances are separated by size due to their different ability to penetrate into the pores of the stationary phase. At the same time, the first of
the largest molecules (with the highest molecular weight) that can penetrate into the minimum number of pores of the stationary phase come out of the columns,
and the substances with small molecular sizes come out last.
Often the separation proceeds not by one, but by several mechanisms at the same time.
The HPLC method can be used to control the quality of any nega-
similar analytes. For analysis, appropriate instruments are used - liquid chromatographs.
The composition of a liquid chromatograph usually includes the following basic
nodes:
– PF preparation unit, including a container with a mobile phase (or a container
sti with individual solvents that are part of the mobile phase
zy) and PF degassing system;
– pumping system;
– mobile phase mixer (if necessary);
– sample injection system (injector);
– chromatographic column (can be installed in a thermostat);
– detector;
– data collection and processing system.
Pumping system
The pumps supply the PF to the column at a given constant rate. The composition of the mobile phase may be constant or variable.
during the analysis. In the first case, the process is called isocratic,
and in the second - gradient. Sometimes installed in front of the pumping system
filters with a pore diameter of 0.45 µm for filtering the mobile phase. Modern
The variable pumping system of a liquid chromatograph consists of one or more pumps controlled by a computer. This allows you to change the
becoming PF according to a specific program with gradient elution. Sme-
the mixing of the PF components in the mixer can occur both at low pressure
ion (before pumps) and at high pressure (after pumps). The mixer can be used for PF preparation and isocratic elution,
however, a more accurate ratio of components is achieved with preliminary
mixing of PF components for an isocratic process. Analytical HPLC pumps make it possible to maintain a constant flow rate of PF into the column in the range from 0.1 to 10 ml/min at a column inlet pressure of up to 50 MPa. It is advisable, however, that this value does not exceed
shalo 20 MPa. Pressure pulsations are minimized by special damping
ferrule systems included in the design of pumps. Working parts on-
pumps are made of corrosion-resistant materials, which allows the use of aggressive components in the composition of the PF.
Faucets
By design, mixers can be static or dynamic.
mic.
In the mixer, a single mobile phase is formed from
specific solvents supplied by pumps, if the required mixture has not been prepared in advance. Mixing of solvents usually occurs spontaneously, but systems with forced mixing are sometimes used.
sewing.
Injectors
Injectors can be universal for introducing samples from
1 µl to 2 ml or discrete for sample injection of only a certain volume
ema. Both types of injectors can be automatic ("auto-injectors" or "auto-samplers"). The injector for entering the sample (solution) is located not -
just before the chromatographic column. The design of the injector makes it possible to change the direction of the PF flow and to preliminarily introduce a sample into a loop of a certain volume (usually from 10 to 100 μl).
This volume is indicated on the loop label. The design of the injector allows the replacement of the loop. To introduce the analyzed solution into non-av-
tomatic injector uses a manual microsyringe with a volume significantly
greatly exceeding the volume of the loop. The excess of the injected solution, not
in the loop is discarded and the exact and always the same volume of sample is injected into the column. Manual incomplete filling of the loop reduces the accuracy
dosing accuracy and reproducibility and, consequently, degrades the accuracy
and reproducibility of chromatographic analysis.
Chromatography column
Chromatographic columns are usually stainless steel, glass or plastic tubes filled with sorbent and closed.
on both sides with filters with a pore diameter of 2–5 µm. The length of the analytical
column, depending on the mechanism of chromatographic separation, can be in the range from 5 to 60 cm or more (usually it is
10-25 cm), inner diameter - from 2 to 10 mm (usually 4.6 mm). Columns with an inner diameter of less than 2 mm are used in microcolumn chromium
tography. Capillary columns with internal diameters are also used.
rum about 0.3-0.7 mm. Columns for preparative chromatography have an internal diameter of up to 50 mm or more.
Before the analytical column, short cables can be installed.
columns (pre-columns) performing various auxiliary functions
(more often - protection of the analytical column). Typically, the analysis is carried out at
at room temperature, however, to increase the separation efficiency and
shortening the duration of the analysis, a thermostat can be used
tirovaniye of columns at temperatures not higher than 60 C. At more than high temperatures possible destruction of the sorbent and a change in the composition of the PF.
Stationary phase (sorbent)
Commonly used sorbents are:
1. Silica gel, alumina, porous graphite are used in normal
small phase chromatography. The retention mechanism in this case
tea - usually adsorption;
2. Resins or polymers with acidic or basic groups. Scope - ion-exchange chromatography;
3. Porous silica gel or polymers (size exclusion chromatography);
4. Chemically modified sorbents (sorbents with grafted fa-
zami), prepared most often on the basis of silica gel. The retention mechanism in most cases is the distribution between mobile
noah and stationary phases;
5. Chemically modified chiral sorbents, for example,
aqueous celluloses and amyloses, proteins and peptides, cyclodextrins,
used to separate enantiomers (chiral chromatography)
Bonded phase sorbents can have varying degrees of chemical
chesky modification. The sorbent particles may be spherical or non-spherical.
regular shape and varied porosity.
The most commonly used bonded phases are:
– octyl groups(sorbent octylsilane or C8);
– octadecyl groups(sorbent octadecylsilane
(ODS) or C18);
– phenyl groups(sorbent phenylsilane);
– cyanopropyl groups(sorbent CN);
– aminopropyl groups(NH2 sorbent);
– diol groups (sorbent diol).
Most often, the analysis is performed on non-polar bonded phases in
reverse-phase mode using C18 sorbent.
In some cases, it is more appropriate to use normal
phase chromatography. In this case, silica gel or polar bonded phases (“CN”, “NH2”, “diol”) are used in combination with non-polar solutes.
Bonded phase sorbents are chemically stable at pH values from 2.0 to 8.0 unless otherwise specified by the manufacturer.
The sorbent particles may have a spherical or irregular shape and a variety of porosity. The particle size of the sorbent in analytical HPLC is usually 3–10 µm, in preparative HPLC, up to 50 µm or more.
Monolithic sorbents are also used.
The high separation efficiency is provided by the high surface area of the sorbent particles (which is a consequence of their microscopy).
the presence of pores), as well as the uniformity of the composition of the sorbent and its dense and uniform packing.
Detectors
Various detection methods are used. In the general case, PF with components dissolved in it after a chromatographic column
ki falls into the detector cell, where one or another of its properties is continuously measured (absorption in the UV or visible region of the spectrum, fluorescence,
refractive index, electrical conductivity, etc.). The resulting chromatogram is a graph of the dependence of some physical
or physico-chemical parameter of the PF on time.
The most common are spectrophotometric
detectors (including diode-matrix), registering a change in the optical
density in the ultraviolet, visible and often in the near infrared
other regions of the spectrum from 190 to 800 or 900 nm. The chromatogram in this case
tea is the dependence of the optical density of the PF on time.
The traditionally used spectrophotometric detector allows
allows detection at any wavelength in its operating range
zone. Multiwave detectors are also used, which allow conducting
perform detection at several wavelengths simultaneously.
With the help of a diode array detector, it is possible not only to carry out detection at several wavelengths at once, but also almost instantly
it is possible (without scanning) to obtain the optical spectrum of the PF at any time, which greatly simplifies the qualitative analysis of the separated components
components.
The sensitivity of fluorescent detectors is approximately 1000 times higher than that of spectrophotometric ones. In this case, either its own fluorescence or the fluorescence of the corresponding derivatives is used, if the analyte itself does not fluoresce. Modern
Interchangeable fluorescent detectors make it possible not only to obtain chromato-
grams, but also to record the excitation and fluorescence spectra of the analy-
zyable connections.
Refractometric detectors are used to analyze samples that do not absorb in the UV and visible regions of the spectrum (for example, carbohydrates).
(refractometers). The disadvantages of these detectors are their low (compared to spectrophotometric detectors) sensitivity and significant temperature dependence of the signal intensity (the detector must be thermostated).
Electrochemical detectors are also used (conductometric
sky, amperometric, etc.), mass spectrometric and Fourier-IR
detectors, detectors of light scattering, radioactivity and some other
mobile phase
AT A variety of solvents, both individual and their mixtures, can be used as PF.
AT normal-phase Chromatography usually uses liquid carbon
hydrocarbons (hexane, cyclohexane, heptane) and other relatively non-polar
solvents with small additions of polar organic compounds,
which regulate the eluting strength of the PF.
In reversed-phase chromatography, the composition of the PF includes polar or-
organic solvents (usually acetonitrile and methanol) and water. For opti-
Separation studies often use aqueous solutions with a certain sign
pH value, in particular buffer solutions. Inorganic additives are used
calic and organic acids, bases and salts and other compounds (on-
example, chiral modifiers to separate enantiomers into achiral-
nom sorbent).
The control of the pH value must be carried out separately for the aqueous component, and not for its mixture with an organic solvent.
PF can consist of one solvent, often two, if necessary
dimity - from three or more. The composition of PF is indicated as the volume ratio of its constituent solvents. In some cases, the mass
ratio, which must be specially stipulated.
When using a UV spectrophotometric detector, the PF should not have a pronounced absorption at the wavelength chosen for detection. The limit of transparency or optical density when determining
the specified wavelength of the solvent of a particular manufacturer is often indicated
is on the package.
The chromatographic analysis is greatly influenced by the degree of purity of the PF, therefore it is preferable to use solvents produced
nye specifically for liquid chromatography (including water).
PF and analyzed solutions should not contain undissolved
particles and gas bubbles. Water obtained in the laboratory
aqueous solutions pre-mixed with water organic solvents
The solvents, as well as the analyzed solutions, must be subjected to fine filtration and degassing. Filtering is usually used for this purpose.
under vacuum through a membrane filter inert with respect to this solvent or solution with a pore size of 0.45 μm.
Data collection and processing system
A modern data processing system is a conjugated
personal computer connected with the chromatograph with software installed
software that allows you to register and process chrono-
matogram, as well as manage the operation of the chromatograph and monitor the main
mi parameters of the chromatographic system.
List of chromatographic conditions to be specified
In a private monograph, the dimensions of the co-
columns, type of sorbent with indication of particle size, column temperature (if temperature control is necessary), volume of injected sample (loop volume),
PF status and method of its preparation, PF feed rate, detector and detection conditions, description of the gradient mode (if used), chromatography time.
ION EXCHANGE AND ION HPLC
Ion exchange chromatography is used for analysis as an organic
skikh (heterocyclic bases, amino acids, proteins, etc.), and non-or-
ganic (various cations and anions) compounds. Separation of components
components of the analyzed mixture in ion-exchange chromatography is based on the reversible interaction of the ions of the analyzed substances with ionic groups.
pami sorbent. Anion exchangers or cation exchangers are used as sorbents.
you. These sorbents are mainly either polymeric ion-
exchange resins (usually copolymers of styrene and divinylbenzene with graft
ionic groups), or silica gels with grafted ion exchange groups. Sorbents with -(CH2)3 N+ X– groups are used to separate anions, and sorbents with -(CH2)SO3 – H+ groups are used to separate cations.
Typically, polymer resins are used to separate anions, and to separate e-
cations are modified silica gels.
As PF in ion-exchange chromatography, aqueous solutions of acids, bases and salts are used. Buffer races are usually used
solutions that allow you to maintain certain pH values. It is also possible to use small additives of water-miscible organic
cal solvents - acetonitrile, methanol, ethanol, tetrahydrofuran.
Ion chromatography- a variant of ion-exchange chromatography, in
which, to determine the concentration of ions of the analyte, is used
using a conductometric detector. For highly sensitive op-
To determine changes in the electrical conductivity passing through the PF detector, the background electrical conductivity of the PF must be low.
There are two main variants of ion chromatography.
The first of them is based on the suppression of the electrical conductivity of the electrolytic
that PF using the second ion-exchange column located between the anal-
lytic column and detector. In this column, neutralization takes place
PF and the analyzed compounds enter the detector cell in deionization
zirovannoy water. The detected ions are the only ions
ensuring the conductivity of the PF. The disadvantage of the suppressor column is the need for its regeneration at fairly short intervals.
me. The suppression column can be replaced by a continuously operating
membrane suppressor, in which the composition of the membrane is continuously
is the flow of regenerating solution moving in the direction
opposite to the direction of the PF flow.
The second version of ion chromatography is single-column ion chromatography.
matography. In this variant, a PF with a very low electrical conductivity is used.
water content. Weak organic compounds are widely used as electrolytes.
sky acids - benzoic, salicylic or isophthalic.
SIZE HPLC
Size exclusion chromatography (gel chromatography) is a special version of HPLC based on the separation of molecules according to their size. Distribution
molecules between the stationary and mobile phases is based on the size of the mo-
molecules and partly on their shape and polarity. For separation, use
porous sorbents - polymers, silica gel, porous glasses and polysaccharides.
The particle size of the sorbents is 5–10 µm.
The advantages of porous glasses and silica gel are fast diffusion of PF and analyte molecules into pores, stability under various conditions (even at high temperatures). Polymeric sorbene-
you are copolymers of styrene and divinylbenzene (this is a hydro-
phobic sorbents used with non-polar mobile phases) and
hydrophilic gels obtained from sulfonated divinylbenzene or polyacrylamide resins.
Two limiting types of interaction of molecules with a porous stationary phase are possible. Molecules larger than the average diameter a pore do not penetrate into the sorbent at all and are eluted together with the mobile phase.
zoy first. Molecules with a diameter much smaller than the pore size of the sort
bents freely penetrate into it, remain in the stationary phase for the longest time and are eluted last. Molecules of medium size penetrate into the pores of the sorbent depending on their size and partially depending on their shape. They elute with different retention times between
our largest and smallest molecules. The separation of the components of the chromatographed sample occurs as a result of repeated ac-
the diffusion of the sample components into the pores of the sorbent, and vice versa.
In size exclusion chromatography, to characterize retention,
a retention volume equal to the product of the PF flow rate and the retention time is used.
mobile phase. The choice of PF depends on the type of sorbent. Exclusion-
chromatography is generally divided into gel filtration and gel chromatography.
permeation chromatography.
The gel filtration chromatography method is used to separate
of water-soluble compounds on hydrophilic sorbents. The mobile phases are aqueous buffer solutions with a given pH value.
Gel permeation chromatography uses hydrophobic
bents and non-polar organic solvents (toluene, dichloromethane, tetra-
hydrofuran). This method is used to analyze compounds that are slightly soluble
rimmed in water.
Detectors. As detectors in size exclusion chromatography, differential refractometric detectors are used, as well as spectrophotometric detectors (including those in the IR region of the spectrum).
Viscometric and flow laser detectors are also used.
These detectors, in combination with a refractometer or other concentration
detector allows you to continuously determine molecular weight on-
limer in PF.
ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY
Ultra performance liquid chromatography is a variant of liquid chromatography that is more efficient
stu compared with classical HPLC.
A feature of ultra-performance liquid chromatography is
the use of sorbents with a particle size of 1.5 to 2 microns. Chro-
matographic columns are usually 50 to 150 mm in length and 1
up to 4 mm in diameter. The volume of the injected sample can be from 1 to 50 µl.
Chromatographic equipment used in the classical
riante HPLC, usually specially adapted for this type of chromatography
Equipment designed for ultra performance liquid chromatography can also be used in the classic version of HPLC.
GENERAL PHARMACOPEIAN AUTHORIZATION
Instead of Art. GF XI
High performance liquid chromatography (high pressure liquid chromatography) is a column chromatography method in which the mobile phase is a liquid moving through a chromatographic column filled with a stationary phase (sorbent). Columns for high performance liquid chromatography are characterized by high hydraulic resistance at the inlet.
Depending on the mechanism of separation of substances, the following variants of high-performance liquid chromatography are distinguished: adsorption, partition, ion-exchange, exclusion, chiral, etc., in accordance with the nature of the main manifested intermolecular interactions. In adsorption chromatography, the separation of substances occurs due to their different ability to be adsorbed and desorbed from the surface of a sorbent with a developed surface, for example, silica gel. In partition high-performance liquid chromatography, separation occurs due to the difference in the distribution coefficients of the substances to be separated between the stationary (as a rule, chemically grafted onto the surface of a stationary carrier) and the mobile phases.
Depending on the type of mobile and stationary phase, normal-phase and reversed-phase chromatography is distinguished. In normal-phase high-performance liquid chromatography, the stationary phase is polar (most often silica gel or silica gel with grafted NH 2 - or CN groups, etc.), and the mobile phase is non-polar (hexane, or mixtures of hexane with more polar organic solvents - chloroform, alcohols, etc.). The retention of substances increases with increasing polarity. In normal phase chromatography, the eluting power of the mobile phase increases with increasing polarity.
In reversed-phase chromatography, the stationary phase is non-polar (hydrophobic silica gels with grafted C4, C8, C18 groups, etc.); the mobile phase is polar (mixtures of water and polar solvents: acetonitrile, methanol, tetrahydrofuran, etc.). The retention of substances increases with the increase in their hydrophobicity (non-polarity). The higher the content of the organic solvent, the higher the eluting power of the mobile phase.
In ion-exchange chromatography, the molecules of the substances of the mixture, dissociated in solution into cations and anions, are separated when moving through the sorbent (cation exchanger or anion exchanger) due to the different strength of the interaction of the ions to be determined with the ionic groups of the sorbent.
In size-exclusion (sieve, gel-penetrating, gel-filtration) chromatography, the molecules of substances are separated by size due to their different ability to penetrate into the pores of the stationary phase. In this case, the largest molecules that can penetrate into the minimum number of pores of the stationary phase are the first to leave the column, and the substances with small molecular sizes are the last to leave.
In chiral chromatography, optically active compounds are separated into individual enantiomers. The separation can be carried out on chiral stationary phases or on achiral stationary phases using chiral mobile phases.
There are other options for high performance liquid chromatography.
separation often proceeds not by one, but by several mechanisms simultaneously, depending on the type of mobile and stationary phases, as well as the nature of the compound being determined.
Application area
High-performance liquid chromatography is successfully used for both qualitative and quantitative analysis of drugs in the tests "Identity", "Foreign impurities", "Dissolution", "Uniformity of dosing", "Quantitative determination". It should be noted that chromatography allows you to combine several tests in one sample, including "Identity" and "Quantification".
Equipment
For analysis, appropriate instruments are used - liquid chromatographs.
The composition of a liquid chromatograph usually includes the following main components:
- a mobile phase preparation unit, including a container with a mobile phase (or containers with individual solvents that are part of the mobile phase) and a system for degassing the mobile phase;
— pumping system;
– mobile phase mixer (if necessary);
– sample injection system (injector), can be manual or automatic (autosampler);
— chromatographic column (can be installed in a thermostat);
— detector (one or more with different detection methods);
— chromatograph control system, data collection and processing.
In addition, the chromatograph may include: a sample preparation system and a pre-column reactor, a column switching system, a post-column reactor, and other equipment.
Pumping system
The pumps supply the mobile phase to the column at a predetermined rate. The composition of the mobile phase and the flow rate can be constant or change during the analysis. In the case of a constant composition of the mobile phase, the process is called isocratic, and in the second - gradient. A modern liquid chromatograph pumping system consists of one or more computer-controlled pumps. This allows you to change the composition of the mobile phase according to a specific program during gradient elution. Pumps for analytical high-performance liquid chromatography allow maintaining the flow rate of the mobile phase into the column in the range from 0.1 to 10 ml/min at a column inlet pressure of up to 40 MPa. Pressure pulsations are minimized by special damper systems included in the design of the pumps. The working parts of the pumps are made of corrosion-resistant materials, which allows the use of aggressive components in the composition of the mobile phase.
Faucets
In the mixer, a single mobile phase is formed from the individual solvents supplied by the pumps, if the required mixture has not been prepared in advance. The mixing of the mobile phase components in the mixer can take place both at low pressure (before the pumps) and at high pressure (after the pumps). The mixer can be used for mobile phase preparation and isocratic elution.
The volume of the mixer can affect the retention time of the components in the gradient elution.
Injectors
Injectors can be universal, with the ability to change the volume of the injected sample, or discrete for introducing a sample of only a certain volume. Both types of injectors can be automatic ("auto-injectors" or "auto-samplers"). The sample injector (solution) is located directly in front of the chromatographic column. The design of the injector makes it possible to change the direction of the flow of the mobile phase and to carry out a preliminary injection of the sample into the dosing loop of a certain volume (usually from 10 to 100 µl) or into a special variable volume dosing device. The volume of the loop is indicated on its marking. The design of a discrete injector, as a rule, allows the replacement of the loop. Modern automatic injectors may have a number of additional features, for example, to perform the function of a sample preparation station: to mix and dilute samples, to carry out a pre-column derivatization reaction.
Chromatography column
Chromatographic columns are usually stainless steel, glass, or plastic tubes filled with a sorbent and closed on both sides with filters with a pore diameter of 2–5 µm. The length of the analytical column can be in the range from 5 to 60 cm or more, the inner diameter is from 2 to 10 mm. Columns with an internal diameter of less than 2 mm are used in microcolumn chromatography. There are also capillary columns with an internal diameter of about 0.3–0.7 mm. Columns for preparative chromatography may have an internal diameter of 50 mm or more.
Before the analytical column, short columns (pre-columns) can be installed that perform various auxiliary functions, the main of which is the protection of the analytical column. Typically, the analysis is carried out at room temperature, however, to increase the efficiency of separation and reduce the duration of the analysis, thermostating of the columns at temperatures up to 80–100 °C can be used. The possibility of using an elevated temperature during separation is limited by the stability of the stationary phase, since its destruction is possible at elevated temperatures.
Stationary phase (sorbent)
Commonly used sorbents are:
- silica gel, alumina, are used in normal phase chromatography. The retention mechanism in this case is usually adsorption;
- silica gel, resins or polymers with grafted acidic or basic groups. Scope - ion-exchange and ion chromatography;
- silica gel or polymers with a given pore size distribution (size exclusion chromatography);
- chemically modified sorbents (bonded phase sorbents), most often prepared on the basis of silica gel. The retention mechanism is adsorption or distribution between the mobile and stationary phases. The scope depends on the type of grafted functional groups. Some types of sorbents can be used in both reversed and normal phase chromatography;
- chemically modified chiral sorbents, for example, cellulose and amylose derivatives, proteins and peptides, cyclodextrins, chitosans used to separate enantiomers (chiral chromatography).
Bonded phase sorbents can have varying degrees of chemical modification. The most commonly used bonded phases are:
– octadecyl groups (sorbent octadecylsilane (ODS) or C 18);
– octyl groups (sorbent octylsilane or C 8);
– phenyl groups (sorbent phenylsilane);
– cyanopropyl groups (CN sorbent);
– aminopropyl groups (NH 2 sorbent);
– diol groups (sorbent diol).
Most often, the analysis is performed on non-polar bonded phases in reverse phase mode using a C 18 sorbent.
Bonded phase sorbents based on silica gel are chemically stable at pH values from 2.0 to 7.0, unless otherwise specified by the manufacturer. The sorbent particles may have a spherical or irregular shape and a variety of porosity. The particle size of the sorbent in analytical high performance liquid chromatography is usually 3–10 µm, in preparative high performance liquid chromatography it is 50 µm or more. There are also monolithic columns in which the sorbent is a monolith with through pores that fills the entire volume of the column.
The high separation efficiency is provided by the high surface area of the sorbent particles (which is a consequence of their microscopic size and the presence of pores), as well as the uniformity of the sorbent composition and its dense and uniform packing.
Detectors
In high performance liquid chromatography, various detection methods are used. In the general case, the mobile phase with the components dissolved in it, after the chromatographic column, enters the detector cell, where one or another of its properties is continuously measured (absorption in the ultraviolet or visible region of the spectrum, fluorescence, refractive index, electrical conductivity, etc.). The resulting chromatogram is a graph of the dependence of some physical or physico-chemical parameter of the mobile phase on time.
The most common detectors in high performance liquid chromatography are spectrophotometric. During the elution of substances in a specially designed microcell, the optical density of the eluate is measured at a preselected wavelength. The wide range of linearity of the detector makes it possible to analyze both impurities and the main components of a mixture on a single chromatogram. A spectrophotometric detector allows detection at any wavelength within its operating range (typically 190-600 nm). Multiwave detectors are also used, which allow detection at several wavelengths simultaneously, and detectors on a diode matrix, which allow recording optical density simultaneously over the entire operating wavelength range (usually 190–950 nm). This makes it possible to record the absorption spectra of the components passing through the detector cell.
The fluorimetric detector is used to detect fluorescent compounds or non-fluorescent compounds in the form of their fluorescent derivatives. The principle of operation of a fluorimetric detector is based on measuring the fluorescent emission of absorbed light. Absorption is usually carried out in the ultraviolet region of the spectrum, the wavelengths of the fluorescent radiation exceed the wavelengths of the absorbed light. Fluorometric detectors have very high sensitivity and selectivity. The sensitivity of fluorescent detectors is approximately 1000 times higher than that of spectrophotometric ones. Modern fluorescent detectors make it possible not only to obtain chromatograms, but also to record the excitation and fluorescence spectra of the analyzed compounds.
To determine compounds that weakly absorb in the ultraviolet and visible regions of the spectrum (for example, carbohydrates), use refractometric detectors (refractometers). The disadvantages of these detectors are their low (compared to spectrophotometric detectors) sensitivity and significant temperature dependence of the signal intensity (the detector must be thermostatted), as well as the impossibility of using them in the gradient elution mode.
Principle of operation evaporative laser light scattering detectors is based on the difference in the vapor pressures of the chromatographic solvents that make up the mobile phase and the analyzed substances. The mobile phase at the outlet of the column is introduced into the nebulizer, mixed with nitrogen or CO 2 and, in the form of a finely dispersed aerosol, enters a heated evaporator tube with a temperature of 30–160 °C, in which the mobile phase evaporates. An aerosol of non-volatile particles of analyzed substances scatters the light flux in the dispersion chamber. By the degree of dispersion of the light flux, one can judge the amount of the determined compound. The detector is more sensitive than the refractometric one, its signal does not depend on the optical properties of the sample, on the type of functional groups in the analytes, on the composition of the mobile phase, and can be used in the gradient elution mode.
Electrochemical detectors (conductometric, amperometric, coulometric, etc.). An amperometric detector is used to detect electroactive compounds that can be oxidized or reduced on the surface of a solid electrode. The analytical signal is the magnitude of the oxidation or reduction current. The detector cell has at least two electrodes - a working electrode and a reference electrode (silver chloride or steel). An operating potential is applied to the electrodes, the value of which depends on the nature of the compounds being determined. Measurements can be carried out both at a constant potential and in a pulsed mode, when the profile of the change in the potential of the working electrode is set during one cycle of signal registration. The amperometric detector uses working electrodes made of carbon materials (most often glassy carbon or graphite), and metal: platinum, gold, copper, nickel.
A conductometric detector is used to detect anions and cations in ion chromatography. The principle of its operation is based on measuring the electrical conductivity of the mobile phase during the elution of a substance.
Exceptionally informative is the mass spectrometric detector, which has high sensitivity and selectivity. The latest models of mass spectrometers for liquid chromatography operate in the m/z mass range from 20 to 4000 amu.
High-performance liquid chromatography also uses Fourier-IR detectors, radioactivity, and some others.
Data collection and processing system
A modern data processing system is a personal computer connected to the chromatograph with installed software that allows you to register and process the chromatogram, as well as control the operation of the chromatograph and monitor the main parameters of the chromatographic system.
mobile phase
The mobile phase in high-performance liquid chromatography performs a dual function: it ensures the transfer of desorbed molecules along the column and regulates the equilibrium constants, and, consequently, retention as a result of interaction with the stationary phase (being sorbed on the surface) and with the molecules of the substances being separated. Thus, by changing the composition of the mobile phase in high performance liquid chromatography, one can influence the retention times of compounds, the selectivity and efficiency of their separation.
The mobile phase may consist of one solvent, often two, if necessary, three or more. The composition of the mobile phase is indicated as the volume ratio of its constituent solvents. In some cases, the mass ratio may be indicated, which should be specially stipulated. Buffer solutions with a certain pH value, various salts, acids and bases, and other modifiers can be used as components of the mobile phase.
Normal phase chromatography usually uses liquid hydrocarbons (hexane, cyclohexane, heptane) and other relatively non-polar solvents with small additions of polar organic compounds that control the eluting strength of the mobile phase.
In reverse phase chromatography, water or aqueous-organic mixtures are used as the mobile phase. Organic additives are usually polar organic solvents (acetonitrile and methanol). To optimize the separation, aqueous solutions with a certain pH value can be used, in particular buffer solutions, as well as various additives to the mobile phase: phosphoric and acetic acids in the separation of acidic compounds; ammonia and aliphatic amines in the separation of basic compounds, and other modifiers.
The purity of the mobile phase greatly affects the chromatographic analysis, so it is preferable to use solvents released specifically for liquid chromatography (including water).
When using a UV spectrophotometric detector, the mobile phase should not have a pronounced absorption at the wavelength chosen for detection. The limit of transparency or optical density at a certain wavelength of a particular manufacturer's solvent is often indicated on the packaging.
The mobile phase and analyzed solutions must be free of undissolved particles and gas bubbles. Water obtained under laboratory conditions, aqueous solutions, organic solvents previously mixed with water, as well as analyzed solutions must be subjected to fine filtration and degassing. For these purposes, vacuum filtration through a membrane filter with a pore size of 0.45 μm, which is inert with respect to a given solvent or solution, is usually used.
List of chromatographic conditions to be specified
The monograph should contain: the full commercial name of the column, indicating the manufacturer and catalog number, column dimensions (length and inner diameter), type of sorbent, indicating the particle size, pore size, column temperature (if temperature control is necessary), volume of the injected sample (volume loops), composition of the mobile phase and method of its preparation, flow rate of the mobile phase, type of detector and detection conditions (if necessary, parameters of the detector cell used), description of the gradient mode (if used), including the stage of re-equilibration to initial conditions, chromatography time, detailed description methods and calculation formulas, descriptions of the preparation of standard and test solutions.
If pre-column derivatization is used in the autosampler, information about the autosampler program is provided. In the case of using post-column derivatization, the feed rate of the derivatizing reagent, the volume of the mixing loop and its temperature are indicated.
Modified High Performance Liquid Chromatography
Ion pair chromatography
One of the varieties of reverse-phase high-performance liquid chromatography is ion pair chromatography - which allows you to determine ionized compounds. For this, hydrophobic organic compounds with ionogenic groups (ion-pair reagents) are added to the traditional reverse-phase high-performance liquid chromatography of the mobile phase. Sodium alkyl sulfates are usually used to separate bases; tetraalkylammonium salts (tetrabutylammonium phosphate, cetyltrimethylammonium bromide, etc.) are used to separate acids. In the ion-pair mode, the selectivity of separation of non-ionic components will be limited by the reversed-phase retention mechanism, while the retention of bases and acids increases markedly, while the shape of the chromatographic peaks improves.
Retention in the ion-pair regime is due to fairly complex equilibrium processes that compete with each other. On the one hand, due to hydrophobic interactions and the effect of displacement of the polar medium of the mobile phase, the sorption of hydrophobic ions on the surface of alkyl silica gel is possible in such a way that the charged groups face the mobile phase. In this case, the surface acquires ion-exchange properties, and retention obeys the laws of ion-exchange chromatography. On the other hand, it is possible to form an ion pair directly in the volume of the eluent, followed by its sorption on the sorbent by the reversed-phase mechanism.
Hydrophilic interaction chromatography ( HILIC chromatography)
Hydrophilic interaction chromatography is used to separate polar compounds that are poorly retained in reverse phase high performance liquid chromatography. As a mobile phase in this variant of chromatography, water-acetonitrile mixtures with the addition of salts, acids or bases are used. The stationary phases, as a rule, are silica gels modified with polar groups (amino, diol, cyanopropyl groups, etc.). More polar compounds are held stronger. The eluting power of the mobile phase increases with increasing polarity.
Ion exchange and ion high performance liquid chromatography
Ion-exchange chromatography is used to analyze both organic (heterocyclic bases, amino acids, proteins, etc.) and inorganic (various cations and anions) compounds. The separation of the components of the analyzed mixture in ion-exchange chromatography is based on the reversible interaction of the ions of the analyzed substances with the ion-exchange groups of the sorbent. These sorbents are mainly either polymeric ion-exchange resins (usually copolymers of styrene and divinylbenzene with grafted ion-exchange groups) or silica gels with grafted ion-exchange groups. Sorbents with groups: -NH 3 +, -R 3 N +, -R 2 HN +, -RH 2 N +, etc. are used to separate anions (anion exchangers), and sorbents with groups: -SO 3 -, -RSO 3 -, -COOH, -PO 3 - and others for the separation of cations (cation exchangers).
As the mobile phase in ion-exchange chromatography, aqueous solutions of acids, bases and salts are used. Typically, buffer solutions are used to maintain certain pH values. It is also possible to use small additions of water-miscible organic solvents - acetonitrile, methanol, ethanol, tetrahydrofuran.
Ion chromatography - a variant of ion-exchange chromatography, in which a conductometric detector is used to detect analytes (ions). For a highly sensitive determination of changes in the conductivity of the mobile phase passing through the detector, the background conductivity of the mobile phase must be low.
There are two main variants of ion chromatography.
The first of these, two-column ion chromatography, is based on the suppression of the electrical conductivity of the mobile phase electrolyte using a second ion-exchange column or a special membrane suppression system located between the analytical column and the detector. When passing through the system, the electrical conductivity of the mobile phase decreases.
The second variant of ion chromatography is single-column ion chromatography. This variant uses a mobile phase with very low electrical conductivity. As electrolytes are widely used weak organic acids: benzoic, salicylic or isophthalic.
Size Exclusion High Performance Liquid Chromatography
Size exclusion chromatography (gel chromatography) is a special version of high performance liquid chromatography based on the separation of molecules by their size. The distribution of molecules between the stationary and mobile phases is based on the size of the molecules and partly on their shape and polarity.
Two limiting types of interaction of molecules with a porous stationary phase are possible. Molecules larger than the maximum pore diameter are not retained at all and are eluted first, moving along with the mobile phase. Molecules with sizes smaller than the minimum pore diameter of the sorbent freely penetrate into the pores and are the last to be eluted from the column. The remaining molecules, which have intermediate sizes, are partially retained in the pores and, during elution, are separated into fractions in accordance with their sizes and, partially, the shape, penetrate into the pores of the sorbent, depending on the size and partially, depending on their shape. As a result, substances are eluted with different retention times.
Ion exclusion chromatography
The mechanism of ion-exclusion chromatography is based on the effect, as a result of which compounds in the ionized form are not retained on the ion-exchange sorbent, while compounds in the molecular form are distributed between the stationary and aqueous phases inside the pores of the ion-exchange sorbent and the mobile phase migrating in the space between the sorbent particles. Separation is based on electrostatic repulsion, polar and hydrophobic interactions between dissolved compounds and the sorbent.
The anionic groups on the surface of the sorbent act as a semi-permeable "membrane" between the stationary and mobile phases. Negatively charged components do not reach the stationary mobile phase, as they are repelled by similarly charged functional groups and eluted in the "dead" (free) volume of the column. Components in molecular form are not "rejected" by the cation-exchange sorbent and are distributed between the stationary and mobile phases. The difference in the degree of retention of the non-ionic components of the mixture is dictated by the combination of polar interactions of non-ionic components with the functional groups of the cation-exchange sorbent and hydrophobic interactions of non-ionic components with the non-polar sorbent matrix.
Chiral chromatography
The goal of chiral chromatography is to separate optical isomers. Separation is carried out on chiral stationary phases or on conventional achiral stationary phases using chiral mobile phases. As chiral stationary phases, sorbents with a modified surface, groups or substances having chiral centers (chitosans, cyclodextrins, polysaccharides, proteins, etc. (chiral selectors) are used. In this case, the same phases can be used as mobile phases as in normal-phase or reversed-phase chromatography.When using achiral stationary phases to ensure the separation of enantiomers, chiral modifiers are added to the mobile phases: chiral metal complexes, neutral chiral ligands, chiral ion-pair reagents, etc.
Ultra performance liquid chromatography
Ultra performance liquid chromatography is a variant of liquid chromatography that is more efficient than classical high performance liquid chromatography.
A feature of ultra-performance liquid chromatography is the use of sorbents with a particle size of 1.5 to 2 µm. Chromatographic columns are typically 50 to 150 mm long and 1 to 4 mm in diameter. The volume of the injected sample can be from 1 to 50 µl. The use of such chromatographic columns can significantly reduce the analysis time and improve the efficiency of chromatographic separation. However, in this case, the pressure on the column can reach 80–120 MPa, the required detector data collection frequency can increase up to 40–100 Hz, and the extracolumn volume of the chromatographic system must be minimized. Chromatography equipment and columns used in ultra high performance liquid chromatography are specially adapted to meet the requirements of this type of chromatography.
Equipment designed for ultra performance liquid chromatography can also be used in classic high performance liquid chromatography.
Introduction
Chromatographic analysis is a criterion for the homogeneity of a substance: if the analyzed substance is not separated by any chromatographic method, then it is considered homogeneous (without impurities).
The fundamental difference between chromatographic methods and other physicochemical methods of analysis is the possibility of separating substances with similar properties. After separation, the components of the analyzed mixture can be identified (set the nature) and quantified (mass, concentration) by any chemical, physical and physico-chemical methods.
Chromatography is widely used in laboratories and industry for the qualitative and quantitative analysis of multicomponent systems, production control, especially in connection with the automation of many processes, as well as for the preparative (including industrial) isolation of individual substances (for example, precious metals), separation of rare and scattered elements.
In accordance with the state of aggregation of the eluent, gas (GC, GC) and liquid chromatography (HPLC, HPLC) are distinguished.
High performance liquid chromatography (HPLC, HPLC) is used to analyze, separate and purify synthetic polymers, drugs, detergents, proteins, hormones and other biologically important compounds. The use of highly sensitive detectors makes it possible to work with very small amounts of substances (10 -11 -10 -9 g), which is extremely important in biological research.
The HPLC method is carried out on various liquid chromatographs. Modern liquid chromatographs are designed to separate complex mixtures of substances into individual components and conduct a qualitative and quantitative analysis of the components of the mixture being separated.
high performance liquid chromatography propyphenazone
In connection with the introduction of GMP into the practice of pharmaceutical production in Russia. the importance of using modern unified methods of analysis is increasing, both at manufacturing enterprises and in the system of state control of the quality of medicines. High performance liquid chromatography (HPLC) is the basic method for analyzing the quality of substances and finished medicinal products in countries with a developed pharmaceutical industry (USA, England, Japan, EU countries). This method, according to its characteristics, meets the requirements of quantitative analysis of about 80-90% of drugs.
The technique for performing any chromatographic determinations is subject to certain General requirements. First of all, it is necessary to note those of them that cause the most questions among beginners.
1. Air conditioning. In the room where the liquid chromatograph is installed, there should be no sharp temperature fluctuations.
Changes in temperature can lead to changes in retention, efficiency, and even separation selectivity.
In summer heat, in unconditioned rooms, it is very difficult to work with normal-phase light-boiling mobile phases. During the day, they gradually evaporate, which leads to a change in the composition of the eluent.
At lower temperatures, problems arise in working with eluents enriched in water and/or containing alcohols. The viscosity of such eluents increases sharply with decreasing temperature, which leads to an increase in pressure in the system.
The influence of small temperature fluctuations on the separation can be eliminated by thermostating the chromatographic column, or the entire liquid system (which is not possible for all chromatographs).
2. Power quality. Most modern chromatographs are equipped with power stabilization systems, however, the quality of the on-site power supply must also be high. If the power supply is not good enough, any run of a series of definitions in automatic mode may fail due to a failure.
3. Purity of solvents. Highly pure solvents should be used for the preparation of mobile phases.
In general, the requirements for the purity of the mobile phase depend on the method of detection, the method of elution (isocratic or gradient), the sensitivity of the detector to the target analyte and its concentration.
When using UV detection, the requirements for the purity of solvents increase with the transition to the short wavelength range, less than 230–240 nm. For isocratic elution with UV detection at wavelengths greater than 220-240 nm, solvents of the "high purity" grade can be used. and distillate water. All reagents added to the mobile phase must also be sufficiently pure; it is useful to recrystallize crystalline reagents before use.
Gradient elution requires "for liquid chromatography" grade solvents and bidistillate water. Special requirements in the gradient elution method (in reverse phase chromatography) are placed on the purity of the aqueous buffer and water in particular. First of all, this is due to the fact that at the initial stage of elution, the adsorbent absorbs polluting components from the mobile phase enriched with an aqueous buffer, which are subsequently eluted and appear on the chromatogram in the form of "humps", "thresholds" and individual peaks, which greatly complicate the extraction of useful analyte signals. .
The purest solvents are required for batch determinations of trace amounts of substances in the gradient elution mode.
To perform determinations in the gradient elution mode, as well as precision determinations in the isocratic mode, the mobile phase must be applied once, that is, the eluate must be discarded or disposed of.
With isocratic elution, if there are no particular sensitivity problems, the spent eluent can be reused. A system in which the eluate, after passing through the detector, is returned to the mobile phase vessel is referred to as a "recycle system". Such a system is especially useful in the case of a large number of routine isocratic determinations on standard columns (250x4.6, 150x4.6) at a flow rate of about 1 ml/min. In these cases, the recycling system saves up to 200-300 ml of organic solvent per day. This economical system allows the use of very pure, expensive solvents for analysis. The issue of saving expensive solvents is less acute in the case of using microcolumns (80x2, 100x2), since separation requires an order of magnitude smaller volume of the mobile phase.
4. Degassing of solvents. Solvents used in chromatography to prepare mobile phases usually contain dissolved air. Especially a lot of air contains water.
When working on non-degassed eluents, air bubbles enter various components of the liquid system: pump, column, capillaries, detector. When air enters the fluid system, high periodic noises appear on the chromatogram, caused by pressure fluctuations in the fluid system. This leads to a sharp decrease in the sensitivity of the analysis.
To remove air from their eluent, it is degassed. As a rule, only eluents for reverse phase separations are degassed - since aqueous-organic mixtures contain significant amounts of dissolved air. Particularly careful degassing must be carried out in the case of gradient elution, as well as when using fluorimetric detection.
In the course of gradient elution in a reversed-phase mode, two eluents are mixed - water-organic mixtures of various compositions. Mixing of non-degassed eluents leads to an intense release of dissolved air, which is critical for the determination as a whole (air bubbles are recorded in the chromatogram as sharp "emissions" on the zero line).
The sensitivity of fluorimetric detection decreases at a high content of dissolved air in the mobile phase (fluorescence is quenched). Thus, when using fluorimetric detection, outgassing of the eluent must be given special attention.
There are three main ways to degas mobile phases for liquid chromatography.
a. Vacuum degassing - the eluent is held in a Claisen flask under water jet pump vacuum for several minutes. During degassing, boiling of the eluent should be avoided.
b. Thermal degassing is used to degas aqueous-organic eluents with a high proportion of water. The mobile phase is placed in a flask, which is not hermetically stoppered, and left in a water bath at a temperature of about 50°C. After 10-15 minutes, the flask is sealed with a stopper and cooled under running water to room temperature.
in. Degassing with ultrasound. The mobile phase is sonicated for several minutes and then allowed to settle for 10-15 minutes. This method is often not efficient enough for the degassing of water-organic eluents.
Modern pumping systems for liquid chromatography are equipped with automatic degassing systems. However, when performing gradient analyses, it is better to degas both mobile phases in advance and "manually", according to one of the above methods.
5. Filtration of the mobile phase. To ensure trouble-free operation of the pump, it is desirable to filter the mobile phase under vacuum using a membrane filter.
6. Washing of the column and components of the liquid system. After working with aqueous organic mobile phases containing salts and acids, the entire liquid system (including the column) should be washed with distilled water with the addition of 5-10% organic solvent. Such flushing is done so that during non-working hours the components of the liquid system of the chromatograph and the stationary phase itself do not wear out additionally.
Failure to carry out such washing leads, first of all, to the fact that after the pump is stopped, salts are deposited from the eluent on its parts and on the walls of the detector cuvette. This, in turn, leads to unstable operation of the device as a whole, as well as premature wear of the moving parts of the pump. Regular failure to flush the system of salt and acid containing eluents can lead to a reduction in the lifetime of the stationary phase.
The addition of some organic solvent to the wash water is necessary to prevent biological contamination of the fluid system.
7. Transition to a new mobile phase that does not mix with the previous one. Such a transition is carried out through an intermediate solvent, which is infinitely miscible with both mobile phases - usually through isopropanol or acetone.
To switch from an aqueous eluent to a nonpolar eluent, the liquid system should be washed with water with the addition of an organic solvent, then the chromatographic column should be removed, the system should be washed with isopropanol (acetone), the system should be washed with a nonpolar eluent, and a new column should be installed.
For the reverse transition, the chromatographic column is removed, the liquid system is washed with isopropanol (acetone), then with an aqueous eluent, and then a new column is installed.
When changing from an aqueous to a non-polar eluent, make sure that the pump seal material is rated for non-polar solvents.
8. Sample filtration. If the analyzed sample contains an undissolved suspension, then it is desirable to filter it by passing the sample through a membrane filter connected to a syringe. Unfortunately, if the sample is small, less than a milliliter, then it becomes almost impossible to filter it in this way.
With regular analysis of samples containing suspended solids, the inlet filter on the column (frit) may become clogged, which will lead, first of all, to an increase in pressure in the system. In this case, it is better to replace the inlet filter, and if there is no replacement, wash it in an organic solvent with sonication for 10-15 minutes.
The most optimal solution to the problem is to apply an in-line filter before the column. The in-line filter contains a replaceable frit - the same as on the column. Replacing the frit on an in-line filter is a routine operation that can be done quite often.
9. Application of guard columns. With the regular analysis of "dirty" samples, the chromatographic column quickly becomes contaminated and loses its separating ability. A well-known alternative to thorough sample preparation in this case is the use of a guard column that protects the main column from contamination.
Sometimes it is expedient not to carry out sample preparation at all, but to put an in-line filter and pre-column in line before the main column. The advantages of this scheme are the simplicity and rapidity of analyzes with less labor and reagents.
10. Preservation of chromatographic columns. Before sufficiently long storage, the chromatographic columns are washed and filled with a solvent that is quite specific for each type of stationary phase.
Thus, chromatographic columns for operation in normal-phase systems are usually filled with high-boiling hydrocarbons, such as isooctane. The reversed phases are washed with water and filled with acetonitrile, or at a low feed rate with isopropanol. Phases intended for operation with aqueous buffers are filled with water with a small addition of sodium azide (bacteriostatic).
Storage instructions for the column may be indicated in its passport.
11. Storage of water buffers. In the case of routine determinations, it can be quite convenient to prepare a large volume of aqueous buffer for the preparation of the mobile phase at once. Unfortunately, an aqueous buffer cannot be stored for more than a few days unless sodium azide, a bacteriostatic, is added to it. Mobile phases based on phosphate buffer are very poorly stored.
Sometimes a large volume of aqueous buffer is prepared in order to "increase the reproducibility of the assay". Generally speaking, with this approach, the reproducibility of the analysis does not increase, but problems with buffer storage appear inevitable.
Generally speaking, the answer to the question is whether to prepare the water buffer for a week or for one day? - is determined solely by the principle of convenience.
12. Regularity of calibration. As a rule, standard calibration is carried out every day, or every time a new eluent is prepared.
Calibration is carried out when the stationary state of the chromatographic system is reached; the readable parameters are the retention time of the standard peak, its area (in case of spectrophotometric detection - at the reference wavelength), spectral ratios (when using a scanning or diode-matrix spectrophotometric detector).
At the beginning of the work, the standard can be analyzed twice - to confirm the reproducibility of the retention time.
1. Determination of the components of the preparation "BICILLIN-3" by HPLC
Bicillin-3 is a long-acting penicillin and is a mixture of sodium, novocaine and benzathine salts of benzylpenicillin (BP). According to the current VFS 42-3034-98, the determination of BP in the preparation is carried out using HPLC, novocaine is determined spectrophotometrically, and benzathine (N,N1-dibenzylethylenediamine) is extracted with ether from an aqueous solution saturated with sodium chloride. After evaporation of the ether, benzathine is determined by titration with perchloric acid.
In the European Pharmacopoeia, the content of BP and benzathine in the benzathine salt of BP is determined using gradient HPLC in a mixture of methanol with a solution of sodium phosphate at pH 3.5.
The purpose of this work is to develop a HPLC method in isocratic mode for the determination of components in bicillin-3.
experimental part
Bicillin-3 produced by AKO Sintez (Kurgan) was used. The study was carried out on a Waters chromatograph (USA) with a model 510 pump, a model 481 UV detector, and a model 7125 injector (Rheodyne) with a dosing loop with a capacity of 50 µL. For detection, a wavelength of 214 nm was used, at which all analyzed compounds are well detected. Registration of chromatograms and calculation of peak areas and main retention parameters was carried out using a personal computer with an analog-to-digital converter and the Multichrome program.
A reverse-phase HPLC method was studied on a Luna C18 (2) column 250 x 4.6 mm from Phenomenex (USA), since the column had previously proven itself to be relatively cheap with improved symmetry of the yield of organic amine peaks. For the same purpose, a mixture of acetonitrile with a buffer solution containing triethylamine as one of the components, having a pH of 5.0, was used as a mobile phase.
The initial solution for the preparation of the mobile phase - 2.5 M solution of phosphoric acid, which was titrated with triethylamine to pH 5.0. An HPLC buffer solution was prepared by diluting the stock solution 10 times with water. 750 ml of the obtained buffer solution was mixed with 250 ml of acetonitrile. At the same time, the apparent pH of the mobile phase increased to 5.7. Mobile phase rate 1 ml/min. Chromatography was carried out at room temperature. Analysis time 20 min.
Since the components of the drug differ in acid-base properties - BP is an acid, and novocaine and benzathine are bases, with an increase in pH, their retention times in the pH range, where their ionization changes, shift in different directions. Therefore, by changing the pH, it is easy to choose a convenient retention of the analyzed components. However, an increase in pH leads to a noticeable deterioration in the shape of the benzathine peak, while a decrease leads to insufficient resolution of novocaine and BP hydrolysis products. The separation of the components of bicillin-3 under the above conditions is shown in the figure. The retention times of novocaine, benzathine, and BP were 4.2, 11.6, and 14.8 min, respectively.
Significant is the output of the peak of novocaine between 2 peaks, which are products of BP hydrolysis. In this regard, for better separation of the components, it is recommended to add small amounts of 2.5 M solutions of phosphoric acid or triethylamine to the mobile phase and control the separation by chromatography of a mixture of novocaine and BP, the solution of which was stored for about a day at room temperature.
For quantitative determination, 20–25 mg of bicillin-3 was added to a 100 ml volumetric flask and dissolved in a 20% aqueous solution of acetonitrile. The use of methanol or its solutions for dissolution led to partial methylation of BP. An increase in the concentration of acetonitrile led to a broadening of the novocaine peak. Upper limit The concentration of a drug is limited by its solubility. Calibration plots for BP and benzathine were obtained using the sodium salt of BP and benzathine diacetate after appropriate conversion. The calibration curve for BP is linear in the region of 0.1-0.5 mg/ml, for benzathine and novocaine - in the region of 0.01-0.05 mg/ml. The results of the determination of the components in 5 series of the drug are presented in table 1, where each value is the average of 5 definitions. The relative standard deviation was 1.6% for novocaine, 3.4% for benzathine and 1.4% for BP.
From Table 1 it follows that the results of quantitative determination using HPLC fit within the allowable limits regulated by ND.
To confirm the correctness of the proposed method, the components of bicillin-3 were analyzed in model mixtures prepared by mixing the sodium salt of BP, benzathine diacetate, and novocaine. The results are shown in table.2. The results are recalculated to the original components.
Each value in the "Found" column of Table 2 is the average result of 3 determinations. The average relative deviation was 2.2% for benzathine, 0.9% for novocaine, and 0.8% for BP, which correlates with the relative mean standard deviations found when analyzing components in real samples. For benzathine, the scatter of the results is somewhat higher than for the other components, which is explained by the low height and irregular shape peak and, accordingly, a larger integration error. Another reason regarding big mistakes in the determination of benzathine, an injector memory effect may occur when analyzing strongly adsorbed substances. However, even such a scatter, slightly exceeding that accepted for analyzes using the HPLC method, is quite acceptable for the determination of benzathine.
conclusions
1. A technique has been developed for the detection and quantitative determination of components in the "Bicillin-3" preparation.
2. The technique has been tested on a number of batches of the drug and confirmed by the analysis of model mixtures of known composition.
2. HPLC in the analysis of preparations containing propyphenazone
Propyphenazone (4-isopropyl-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one; isopropylantipyrine) belongs to non-narcotic analgesics of the pyrazolone series and is a part of combined over-the-counter drugs. Currently, caffetin tablets are widely used in medical practice (composition: propyphenazone-0.21 g, paracetamol-0.25 g, caffeine-0.05 g, codeine phosphate-0.01 g) and saridon (paracetamol-0.25 g, propyphenazone-0.15 g, caffeine-0.05 g). According to the existing regulatory documentation, chromatography in a thin layer of a sorbent is used to confirm the authenticity of caffetin tablets. Quantitative determination is proposed to be carried out in separate weighed portions of the drug using methods different for each component: using spectrophotometry, titrimetry, a combination of thin layer chromatography and spectrophotometry. In the regulatory documentation for saridon, paracetamol, caffeine and propyphenazone are determined by HPLC on columns 12.5 cm long with a Merck Lichrospher C18 sorbent and a mobile phase of the composition: methanol-0.01 M phosphoric acid in a ratio of 30:70.
The purpose of this work is to develop methods for detecting and quantifying the components of caffetin and saridon tablets using HPLC.
We used tablets of caffetine produced by Alkaloid Skopje (Republic of Macedonia), saridon produced by Roche Nicholas S.A. Laboratories, Gaillard (France) and the substances of their components. The study was carried out on a domestic microcolumn liquid chromatograph "Milichrom-4" with a UV spectrophotometric detector, a column 8 cm long with a reverse-phase sorbent Separon-C18 as a stationary phase. The polar nature of the analyzed compounds, their good solubility in water and acetonitrile led to the choice of water-acetonitrile mixtures in different ratios as the mobile phase. Mobile phases were tested acetonitrile - water in ratios of 9: 1; 7:3; 6:4; 8:2 and acetonitrile-water-diethylamine (3:2:0.2). The use of mobile phases with a volume fraction of an organic solvent of more than 80% was avoided in order to exclude normal-phase interactions that hinder further control of the composition of the mobile phase. A solvent volume fraction of less than 5% leads to functional instability of the mobile phase and irreproducibility of retention times. The introduction of diethylamine as a modifier into the composition of the mobile phase made it possible to separate all 4 components of caffetin tablets. It is known that on the surface of octadecyl silica gel there is a significant amount of residual silanol groups capable of ion-exchange interaction. Diethylamine eliminates silanol groups from the chromatographic process, improves peak shape, reduces analysis time, and regulates the pH on the silica gel surface. The measurement was carried out under the following conditions: registration scale 2.0, retention time 0.8 s, eluent flow rate 50 μl/min, sample injection volume 3 μl. Peak detection was carried out at 2 wavelengths - 238 and 276 nm.
Identification was carried out according to the retention parameters, which were previously determined on standard solutions of the studied substances.
The components of caffetin tablets are separated using an acetonitrile-water-diethylamine (3:2.2:0.2) mobile phase. The retention time of paracetamol was 3.08 minutes, propyphenazone - 5.73 minutes, caffeine - 4.0 minutes, codeine - 4.67 minutes.
The components of saridon can also be separated using an acetonitrile-water (8:2) mobile phase. Retention time for paracetamol - 3.9 min, propyphenazone - 5.11 min, caffeine - 4.44 min.
An absolute calibration method was used for quantification. A direct proportional dependence of the concentration of the substance on the height of the peak was observed for paracetamol in the range of 50-200 μg / ml, for propyphenazone - 25-128 μg / ml, caffeine - 20-50 μg / ml, codeine - 59-234 μg / ml.
The HPLC method has some limitations in the analysis of complex mixtures. With the simultaneous presence of substances in the mixture in macro- and microquantities, column overload occurs, which affects the quality of separation and the shape of the outgoing peaks. In caffetine, the content of codeine phosphate in relation to paracetamol and propyphenazone is 21-25 times less, therefore liquid extraction is recommended to separate codeine from the rest of the tablet components. Previously, we found that paracetamol, propyphenazone and caffeine are extracted with a single extraction with ethyl acetate from aqueous solutions at pH 2.0 in the amount of 87.43, 87.29 and 87.84%, respectively, and codeine remains completely in the aqueous solution and for its extraction and concentration it is necessary to use chloroform at pH 9.0-10.0.
Method for the quantitative determination of paracetamol, propyphenazone and caffeine in tablets of saridon and caffetin. 20 tablets are ground in a mortar into a fine homogeneous powder, about 0.01 g (accurately weighed) of the powder of the crushed tablets is weighed and placed in a volumetric flask with a capacity of 25 ml, 10 ml of acetonitrile are added and mix thoroughly.
The contents of the flask were made up to the mark with acetonitrile, mixed and filtered. The solution is injected into the chromatograph column in a volume of 3.0 μl. The content of paracetamol, propyphenazone and caffeine is determined by the method of absolute calibration. The results of the determination are shown in Tables 1 and 2, from which it can be seen that the data obtained are within the allowable limits of the content according to the normative documentation (RD).
Method for the quantitative determination of codeine phosphate in caffetin tablets. About 0.2 g of crushed tablets (accurately weighed) are dissolved in 20 ml of water, mixed well until a homogeneous solution is obtained, filtered through a filter for fine and very fine sediments, the filter is washed with 10 ml of purified water. The solution is acidified with 10% sulfuric acid to pH 2.0. Extract three times with 10 ml portions of ethyl acetate. The extracts are discarded. A 25% ammonia solution is added to the aqueous solution to pH 9.0-10.0. Extracted three times with chloroform in portions of 10 ml. The combined chloroform extracts are placed in porcelain dishes and evaporated at room temperature. Dry residues are dissolved in acetonitrile, transferred to a 25 ml volumetric flask and made up to the mark with the same solvent. 3 μl of the resulting solution is injected into a chromatograph column and codeine is determined under the described conditions. The results of the determination are shown in table.3.
To assess the accuracy of the proposed methods and check the reproducibility of the results, model mixtures were prepared and studied. Data on the example of saridon tablets are given in table.4. As can be seen from Table 4, the relative error of determination does not exceed ±1.19% for paracetamol, ±1.16% for propyphenazone, and ±1.63% for caffeine.
Method for the quantitative determination of the components of saridon tablets in model mixtures. Accurate weights of paracetamol (about 0.08 g), propyphenazone (about 0.05 g) and caffeine (about 0.016 g) are weighed out, transferred to a 50 ml volumetric flask, dissolved in a small volume of acetonitrile and adjusted to the mark with the same solvent. A 2.5 ml aliquot is taken and transferred to a 25 ml volumetric flask, the volume is adjusted to the mark with the same solvent, mixed and filtered. The solution is injected into the chromatograph column in a volume of 3 µl.
conclusions
1. A technique for detecting the components of caffetin and saridon tablets using HPLC has been developed.
The retention time for paracetamol was 3.08 minutes, for propyphenazone 5.73 minutes, caffeine 4 minutes and codeine 4.67 minutes.
2. An HPLC method was proposed for the quantitative determination of the components of caffetin and saridon tablets.
The relative error of determination was ±1.19-1.21% for paracetamol, ±1.16-1.71% for propyphenazone, ±1.22-1.63% for caffeine, and ±2.95% for codeine.
3. Standardization of the drug "Adanol"
Pharmaceutical company "Polisan" has developed a number of complex metabolic drugs that stimulate the metabolic processes of the brain, including "Cytoflavin" (injections, tablets) and "Adanol". "Adanol" has pronounced antihypoxic and anti-ischemic properties and is promising medicine for the treatment of patients with the consequences of a stroke. It is a tablet dosage form coated with an enteric coating.
It consists of succinic acid (YA), piracetam (Pc), riboxin (Rb), nicotinamide (NA), pyridoxine hydrochloride (PG), riboflavin mononucleotide (RF).
The purpose of the work is to develop a method for the qualitative and quantitative determination of UC in complex multicomponent mixtures using the example of the drug "Adanol".
We used a high-pressure liquid chromatograph from Shimadzu (Japan) with a UV detector and a Hypersil BDS C18 column from Supelco Inc. grain size 5 µm, length 250 mm, inner diameter 4.6 mm. The mobile phase is a water-organic phase based on a phosphate buffer (pH 2.6-7.0). The detection wavelength is 206 nm. Analysis mode isocratic, elution rate 500 µl/min; sample volume 20 µl. UV spectra were recorded on a Shimadzu UV mini-1240 spectrophotometer.
For the quantitative determination of most of the substances that make up the drug, spectrophotometric methods of analysis have been proposed. However, a comparison of the spectral characteristics of the components of the preparation showed that the absorption regions of YA, PG, NA, Pb, and Pc in the UV zone overlap each other (Fig. 1).
In this regard, their content in the mixture cannot be determined by direct spectrophotometry, and in this case it is advisable to use the HPLC method. Only RF has a specific absorption region of more than 350 nm (lmax=373, E1% 1cm=202 and lmax=445 nm, E1% 1cm=243), therefore, a qualitative and quantitative analysis by the spectrophotometric method is proposed for it.
On the basis of the data obtained, the optimal working wavelength for the analysis of 5 substances was chosen, which is lopt = 206 nm (see Fig. 1). This value is the maximum of the UV spectrum of UC, which has the lowest specific absorption (lmax=206 nm E1% 1cm=5.8) compared to the other components of the mixture (see table).
Since all substances included in the preparation are ionogenic, it is advisable to use reversed-phase chromatography for their analysis using non-polar stationary and polar mobile phases. In reverse-phase chromatography of ionic compounds, the pH value is one of the factors that significantly affects the efficiency of separation of individual substances. When working with modern reverse-phase sorbents, buffer solutions pH from 2.0 to 8.0 are usually used as part of the mobile phase. Since the selected optimum operating wavelength is 206 nm, it is best to use a phosphate buffer because it has no position in the UV wavelength range above 200 nm.
In order to study the behavior of the YaC at different values Its pH spectra were taken in phosphate buffer solutions pH 7.0 and 2.6 (Fig. 2). At pH 2.6, a hypochromic effect of the molecular form of UC is observed - absorption decreases by a factor of 3 compared with the spectrum at pH 7.0 (at this pH value, the dissociation of UC is completely suppressed). With this in mind, the optimal pH value of the mobile phase is 7.0. Further, the influence of the composition and pH of the mobile phase on the efficiency of separation of the drug components was studied. At pH 2.6, the mixture did not completely separate into individual peaks - Pc, Na and PG do not separate and come out as the first one peak, followed by YA and Pb in the form of individual peaks. At pH 5.5 there was also no complete separation of the components. At pH 7.0 there was a complete separation of the components of the mixture. All substances came out as individual peaks in the following sequence: YA, Pc, PG, NA, and Pb. However, the separation process is lengthy - 45-50 minutes.
To ensure a greater eluting power of the mobile phase and accelerate the separation process, it is necessary to introduce a less polar organic solvent into its composition. Of the solvents most commonly used as eluting agents in HPLC, we could use acetonitrile and methanol in terms of the transparency limit in UV light at the chosen working wavelength (lopt = 206 nm), whose transparency limits are 195 and 205 nm, respectively.
The introduction of 2% methanol into the composition of the mobile phase reduced the process time, however, the HA peak had a high asymmetry, and the Pb peak was superimposed on the tail of the HA peak. After reducing the methanol concentration to 1%, the Pb and HA peaks did not overlap, but the asymmetry of the HA peak increased. To reduce it, acetonitrile was introduced into the mobile phase containing 1% methanol.
As a result of the experiments, a mobile phase was selected - an aqueous-organic phase consisting of a phosphate buffer pH 7.0 containing 1% methanol and 0.5% acetonitrile, which ensured the effective separation of all 5 components (Fig. 3). The total chromatography time in this system was 35 min, and the retention times of the components were approximately (in min): 5.3 for YaK, 15 for Pc, 19.3 for PG, 26 for NA, and 31 for Pb.
Quantitative determination was carried out by the external standard method using solutions of standard samples of individual components.
The metrological characteristics of the proposed method of quantitative determination were studied on model mixtures in 5 replications and are presented in the table.
As follows from the table, using the developed method in the preparation "Adanol" it is possible to determine all 5 components with a relative error of no more than 3% with confidence level 95%.
In addition to the peaks of the main components of the drug, the chromatogram obtained under the above conditions revealed peaks of hypoxanthine and nicotinic acid present in the initial substances, as well as those formed during hydrolysis from Pb and NA, respectively. Thus, the developed technique makes it possible to qualitatively and quantitatively determine these foreign impurities in the preparation.
conclusions
1. The optimal conditions for the quantitative analysis of succinic acid using the HPLC method in a multicomponent mixture were determined.
2. A technique has been developed for the qualitative and quantitative analysis of the components of the drug "Adanol", including impurities, with a relative error of determination of no more than 3%.
List of used literature
1. M.A. Kazmin, A.V. Mikhalev, A.P. Arzamastsev "Determination of the components of the drug "BICILLIN-3" by HPLC" // Pharmacy - No. 5 - 2002 - p.5-6.
2. T.Kh. Vergeichik, N.S. Onegov "HPLC in the analysis of preparations containing propyphenazone" // Pharmacy - No. 6 - 2002 - p.13-16.
3. A.Yu. Petrov, S.A. Dmitrichenko, A.L. Kovalenko, L.E. Alekseeva "Standardization of the drug "Adanol" // Pharmacy - No. 5 - 2002 - p.11-13.
Additional
1. Baram G.I., Fedorova G.A. // Application of chromatography in the food, microbiological and medical industries: Mat. Vses. Conf. - Gelendzhik, 1990 - S.43-44.
2. Krichkovskaya L.V., Chernenkaya L.A. // Application of chromatography in the food, microbiological and medical industries: Mat. Vses. Conf. - Gelendzhik, October 8-12, 1990, M., 1990. - P.49.
3. Chromatography: Practical application of the method: In 2 parts. Part 2 - M.: Mir, 1986. - 422 p.