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HPLC chromatography. High performance liquid chromatography (HPLC). Liquid adsorption chromatography column

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HPLC is a liquid column chromatography in which a variety of sorption mechanisms can be used. Essentially, HPLC is a modern form of classical liquid column chromatography. Some of the most significant quality characteristics of HPLC are listed below:
- high speed of the process, which made it possible to reduce the duration of separation from several hours and days to minutes;
- minimal degree of blurring of chromatographic zones, which makes it possible to separate compounds that only slightly differ in sorption constants;
- a high degree of mechanization and automation of information separation and processing, thanks to which column liquid chromatography has reached a new level of reproducibility and accuracy.

Intensive research in recent decades and the enormous amount of accumulated experimental data now allow us to talk about the classification of variants within the framework of the high-performance liquid chromatography method. Of course, the classification according to the sorption mechanism given above remains valid.

A common classification is based on the comparative polarity of the mobile and stationary phases. In this case, a distinction is made between normal and reverse phase chromatography.

Normal phase chromatography (NPC) is a variant of HPLC when the mobile phase is less polar than the stationary phase, and there is reason to believe that the main factor determining retention is the interaction of sorbates directly with the surface or volume of the sorbent.

Reversed phase chromatography (RPC) is a variant of HPLC where the mobile phase is more polar than the stationary phase, and retention is determined by direct contact of sorbate molecules with the surface or volume of the sorbent; in this case, ionized sorbates are not exchanged for mobile phase ions sorbed on the surface.

Ion exchange chromatography is an option in which sorption is carried out by exchanging sorbed ions of the mobile phase for ions of the substances being chromatographed; Ligand exchange chromatography can be defined in a completely analogous manner.

Chromatography on dynamically modified sorbents is a variant of HPLC in which the sorbate does not interact directly with the surface of the sorbent, but enters into association with molecules of the surface layers of the eluent.
Ion-pair chromatography is a variant of reverse-phase chromatography of ionized compounds in which a hydrophobic counterion is added to the mobile phase, which qualitatively changes the sorption characteristics of the system.

Size exclusion chromatography is a method of separating compounds by their molecular weights, based on the difference in the diffusion rate of molecules of different sizes in the pores of the stationary phase.

For HPLC, a very important characteristic is the size of the sorbents, usually 3-5 microns, now up to 1.8 microns. This allows complex mixtures of substances to be separated quickly and completely (average analysis time from 3 to 30 minutes).

The separation problem is solved using a chromatographic column, which is a tube filled with a sorbent. When performing an analysis, a liquid (eluent) of a certain composition is fed through a chromatographic column at a constant speed. A precisely measured dose of sample is injected into this stream. The components of a sample introduced into a chromatographic column, due to their different affinities for the column sorbent, move along it at different speeds and reach the detector sequentially at different times.

Thus, the chromatographic column is responsible for the selectivity and efficiency of separation of components. By selecting different types of columns, the degree of separation of the analytes can be controlled. Compounds are identified by their retention time. Quantitative determination of each of the components is calculated based on the magnitude of the analytical signal measured using a detector connected to the output of the chromatographic column.

Sorbents. The development of HPLC is largely associated with the creation of new generations of sorbents with good kinetic properties and diverse thermodynamic properties. The main material for sorbents in HPLC is silica gel. It is mechanically strong and has significant porosity, which gives a large exchange capacity with small column sizes. The most common particle size is 5-10 microns. The closer to the spherical shape of the particles, the lower the flow resistance, the higher the efficiency, especially if a very narrow fraction is screened out (for example, 7 +1 microns).

The specific surface of silica gel is 10-600 m /g. Silica gel can be modified with various chemical groups grafted to the surface (C-18, CN, NH2, SO3H), which allows the use of sorbents based on it to separate a wide variety of classes of compounds. The main disadvantage of silica gel is its low chemical resistance at pH< 2 и рН >9 (silica dissolves in alkalis and acids). Therefore, there is currently an intensive search for sorbents based on polymers that are stable at pH from 1 to 14, for example, based on polymethyl methacrylate, polystyrene, etc.

Sorbents for ion exchange chromatography. Due to the peculiarities of separation (in an acidic or alkaline environment), the main material is sorbent into polystyrene with divinylbenzene of varying degrees of cross-linking with SO3 -H+ (strongly acidic cation exchangers) or -COO-Naf (weakly acidic cation exchangers), -H2N+ (CH3) groups grafted to their surface 3Cl- (strong basic anion exchangers) or -N+HR2Cl- (weak basic anion exchangers).

Sorbents for gel permeation chromatography. The main type is styrene-DVB. Macroporous glasses, methyl methacrylate, and silica gel are also used. The same sorbents are used for ion exclusion chromatography.
Pumps. To ensure the flow of the mobile phase (MP) through the column with the specified parameters, high-pressure pumps are used. The most important technical characteristics of LC pumps include: flow range; maximum working pressure; flow reproducibility; solvent supply pulsation range.

Depending on the nature of the solvent supply, pumps can be of constant supply (flow) and constant pressure. Basically, during analytical work, a constant flow rate mode is used, and when filling columns, a constant pressure mode is used. Based on their operating principle, pumps are divided into syringe pumps and reciprocating plunger pumps.

Syringe pumps. Pumps of this type are characterized by an almost complete absence of pulsations in the flow of the mobile phase during operation. Disadvantages of the pump: a) high consumption of time and solvent for washing when changing the solvent; b) suspension of separation while filling the pump; c) large dimensions and weight while ensuring high flow and pressure (a powerful engine and a large piston force with its large area are needed).

Reciprocating plunger pumps. Pumps of this type provide a constant volumetric supply of the mobile phase for a long time. Maximum operating pressure 300-500 atm, flow rate 0.01-10 ml/min. Volume flow reproducibility is 0.5%. The main disadvantage is that the solvent is supplied to the system in the form of a series of successive pulses, so there are pressure and flow pulsations.

This is the main reason for the increased noise and reduced sensitivity of almost all detectors used in LC, especially electrochemical ones. Ways to combat pulsations: using double pumps or a double-plunger Bag-Lai pump, using damping devices and electronic devices.

The amount of volumetric feed is determined by three parameters: the diameter of the plunger (usually 3.13; 5.0; 7.0 mm), its amplitude (12-18 mm) and frequency (which depends on the rotation speed of the motor and gearbox).

Dispensers. The purpose of the dispenser is to transfer a sample at atmospheric pressure to the inlet of a column at a pressure of up to several atmospheres. It is important that there are no “dead” volumes in the dispenser that cannot be washed by the mobile phase and that there is no erosion of the sample during dosing. At first, LC dispensers were similar to gas dispensers with a puncture of the membrane. However, the membranes do not withstand more than 50-100 atm; their chemical resistance is insufficient; their pieces contaminate column filters and capillaries.

The liquid phase has a much lower diffusion rate than the gas phase. Therefore, you can dose by stopping the flow - the sample does not have time to erode in the dispenser. While the sample is being introduced into the dispenser, a special valve shuts off the solvent flow. The pressure at the inlet to the column decreases quickly; after a few seconds, the sample can be injected into the dispenser chamber with a conventional microsyringe. Next, the dispenser is locked, the solvent flow is turned on, and separation occurs.

The pressure that this tap holds is up to 500-800 atm. But when the flow stops, the equilibrium in the column is disturbed, which can lead to the appearance of “vacant” additional peaks.

Loop dispensers are the most widely used. When the dispenser is filled, inlets 1, 2 and the channel between them are under high pressure. Inputs 3-6, the channels between them and the dosing loop are under atmospheric pressure, which allows you to fill the loop using a syringe or pump. When the dispenser is turned, the flow of the mobile phase displaces the sample into the column. To reduce the error, the loop is washed with 5-10 times the sample volume. If the sample is small, it can be injected into the loop with a microsyringe. The loop volume is usually 5-50 µl.

ON THE. Voinov, T.G. Volova

Liquid adsorption chromatography column

The separation of a mixture of substances in an adsorption column occurs as a result of their difference in sorption 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 internal surface that retain liquids using 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 following requirements are imposed on adsorbents: S they must not enter into chemical reactions with the mobile phase and separated substances; S must have mechanical strength; S adsorbent grains must be of the same degree of dispersion.

When choosing conditions for the chromatographic process, the properties of the adsorbent and adsorbed substances are taken into account.

In the classic version of liquid column chromatography (LCC), an eluent (SF) is passed through a chromatographic column, which is a glass tube with a diameter of 0.5 - 5 cm and a length of 20 - 100 cm, filled with a sorbent (SF). The eluent moves under the influence of gravity. The speed of its movement can be adjusted using the tap located 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 selected, which are analyzed by some method that allows measuring the concentrations of the substances being determined.

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. for preparation for analysis by other methods (including chromatographic). For example, a mixture of tocopherols is separated on an alumina column, the eluent is passed through, and the a-tocopherol fraction is collected for subsequent determination by the photometric method.

Chromatographic separation of a mixture on a column due to the slow progress of the PF takes a lot of time. To speed up the process, chromatography is carried out under pressure. This method is called high-performance liquid chromatography (HPLC)

Modernization of equipment used in classical liquid column chromatography has made it one of the most promising and modern methods of 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 reverse-phase high-performance liquid chromatography (RPHLC).

During the transition from eluent to eluent, equilibrium under HPLC conditions is established many times faster than under conditions of polar sorbents and non-aqueous PFs. As a result of this, as well as the convenience of working with aqueous and aqueous-alcohol eluents, OFVLC has now gained great popularity. Most HPLC analyzes are carried out using this method.

Equipment 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 the eluent according to a specific program. The pumps create pressure up to 40 MPa. The sample is introduced 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 a microcomputer 11. If necessary, fractions are automatically selected at the moment the peak appears.

Columns for HPLC are made of stainless steel with an internal diameter of 2–6 mm and a length of 10–25 cm. The columns are filled with sorbent (SF). Silica gel, aluminum oxide or modified sorbents are used as NF. Silica gel is usually modified by chemically introducing various functional groups into its surface.

Detectors. The output of an individual component from the column is recorded using a detector. For registration, you can use a change in any analytical signal coming from the mobile phase and associated with the nature and quantity of the mixture component. Liquid chromatography uses analytical signals such as light absorption or light emission of the output solution (photometric and fluorimetric detectors), refractive index (refractometric detectors), potential and electrical conductivity (electrochemical detectors), etc.

The continuously detected signal is recorded by a recorder. A chromatogram is a sequence of detector signals recorded on a recorder tape, generated when individual components of a mixture leave the column. If the mixture is separated, individual peaks are visible on the external chromatogram. The position of the peak on the chromatogram is used for the purpose of identifying the substance, the height or area of the peak - for the purpose of quantitative determination.

Qualitative analysis

The most important characteristics of the chromatogram - retention time tR and the associated retained volume - reflect the nature of the substances, their ability to sorption on the stationary phase material and, therefore, under constant chromatography conditions, 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 (Fig. 7.1.1.2), where t.R(A) is the retention time of component A of the analyzed mixture from the moment of entry into the column until the maximum peak appears at the exit from the column, 1K( hs) is the retention time of the internal standard (the substance initially absent from the analyzed mixture), h is the peak height (mm), ash is the peak width at half its height, mm.

To identify a substance from a chromatogram, standard samples or pure substances are usually used. Compare the retention time of the unknown component IR* with the retention time IRCT of known substances. But identification is more reliable 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 mixture under study is chromatographically separated (chromatographed), to which the internal standard is first 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, it is preferable to measure h, for wide blurred peaks - S. The peak area is measured in different ways: by multiplying the height of the peak (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.

To determine the content of substances in a sample, mainly three methods are used: the absolute calibration method, the internal normalization method and the internal standard method.

The absolute calibration method is based on the preliminary determination of the relationship between the amount of the introduced substance and the area or height of the peak in the chromatogram. A known amount of the calibration mixture is introduced into the chromatogram and the areas or heights of the resulting peaks are determined. Construct a graph of the dependence of the peak area or height on the amount of the administered substance. The test sample is analyzed, the area or height of the peak of the component being determined is measured, and its quantity is calculated based on the calibration graph.

This method provides information only about the relative content of the component in the mixture, but does not allow one to determine its absolute value.

The internal standard method is based on comparing a selected peak parameter of the 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 quite well separated from the peaks of the components of the test mixture.

The last two methods require the introduction of correction factors that characterize 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 solution fractions collected at the moment the substance leaves the column. The analysis can be carried out using 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. Using HPLC, you can determine dyes, organic acids, amino acids, sugars, impurities of pesticides and herbicides, medicinal substances and other pollutants in food products.

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 only difference is in the use of liquid as a stationary phase. Due to the high density of liquid mobile phases and the high resistance of columns, gas and liquid chromatography differ greatly in instrumentation.

In HPLC, pure solvents or mixtures thereof are usually used as mobile phases.

To create a stream of pure solvent (or mixtures of solvents), called an eluent in liquid chromatography, pumps included in the hydraulic system of the chromatograph are used.

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 during movement with the mobile phase along the column. The zone separation of the components achieved in this case depends on the interaction with both the solvent and the adsorbent.

The most widely used in HPLC are silica gel adsorbents with different volumes, surface areas and pore diameters. 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 high pressures characteristic of HPLC;

silica gel, compared to aluminum oxide, has a wider range of porosity, surface area and pore diameter; The significantly greater catalytic activity of aluminum oxide leads to distortion of analysis results due to the decomposition of sample components or their irreversible chemisorption.

Detectors for HPLC

High-performance liquid chromatography (HPLC) is used to detect polar non-volatile substances that, for some reason, cannot be converted into a form suitable for gas chromatography, even in the form of derivatives. Such substances, in particular, include sulfonic acids, water-soluble dyes and some pesticides, for example phenyl-urea derivatives.

Detectors:

UV detector on a diode matrix. A “matrix” of photodiodes (more than two hundred of them) constantly registers signals in the UV and visible regions of the spectrum, thus providing recording of UV-B spectra in scanning mode. This allows you to continuously record, at high sensitivity, undistorted spectra of components quickly passing through a special cell.

Compared to single wavelength detection, which does not provide information about peak purity, the ability to compare full spectra of a diode array provides a much higher degree of confidence in the identification result.

Fluorescence detector. The great popularity of fluorescent detectors is due to their very high selectivity and sensitivity, and the fact that many environmental pollutants fluoresce (eg polyaromatic hydrocarbons).

Electrochemical detector used to detect substances that are easily oxidized or reduced: phenols, mercaptans, amines, aromatic nitro- and halogen derivatives, aldehydes, ketones, benzidines.

Detectors. The output of an individual component from the column is recorded using a detector. For registration, you can use a change in any analytical signal coming from the mobile phase and associated with the nature and quantity of the mixture component. Liquid chromatography uses analytical signals such as light absorption or light emission of the output solution (photometric and fluorimetric detectors), refractive index (refractometric detectors), potential and electrical conductivity (electrochemical detectors), etc.

The continuously detected signal is recorded by a recorder. A chromatogram is a sequence of detector signals recorded on a recorder tape, generated when individual components of a mixture leave the column. If the mixture is separated, individual peaks are visible on the external chromatogram. The position of the peak in the chromatogram is used for the purpose of identifying the substance, the height or area of the peak - for the purpose of quantitative determination.

“High performance liquid chromatography of pollutants in natural and waste waters”

Introduction

Chapter 1. Basic concepts and classification of liquid chromatography methods

1.1 Equipment for liquid chromatography

Chapter 2. The essence of HPLC

2.1 Application

Chapter 3. Examples of the use of HPLC in the analysis of environmental objects

Chapter 4. HPLC equipment

Literature

Application

Introduction

Chromatographic methods often prove indispensable for the identification and quantification of organic substances with similar structures. However, gas chromatography and high performance liquid chromatography are the most widely used for routine analysis of environmental pollutants. Gas chromatographic analysis of organic pollutants in drinking water and wastewater was initially based on the use of packed columns, and later quartz capillary columns became widespread. The internal diameter of capillary columns is usually 0.20-0.75 mm, length - 30-105 m. Optimal results when analyzing pollutants in water are most often achieved when using capillary columns with different film thicknesses of methyl phenyl silicones with a phenyl group content of 5 and 50% . The weak point of chromatographic techniques using capillary columns is often the sample introduction system. Sample introduction systems can be divided into two groups: universal and selective. Universal ones include split and splitless injection systems, “cold” injection into the column and evaporation with temperature programming. When selective input is used, purging with intermediate capture in a trap, 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 entering 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, which respond to each component in the mobile phase, and selective, which respond to the presence in the mobile phase of a certain group of substances with similar chemical characteristics. Universal detectors 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 atoms of halogens, sulfur and nitrogen). Minimum detectable amounts of substances range from nanograms to picograms per second.

High performance liquid chromatography(HPLC) is an ideal method for the determination of a large number of thermally labile compounds that cannot be analyzed by gas chromatography. Modern agrochemicals, which include methyl carbonates and organophosphorus insecticides, and other non-volatile substances, are often the objects of analysis using liquid chromatography. High-performance liquid chromatography is becoming increasingly widespread among other methods used in environmental monitoring, also because it has brilliant prospects in terms of automation of 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 carrier. The simple instrumentation of paper and thin-layer chromatography has led to the widespread use of these methods in analytical practice. However, the great capabilities 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 dramatically increase the speed of analysis and significantly increase the separation efficiency due to the use of finely dispersed sorbent. The HPLC method currently allows the isolation, quantitative and qualitative analysis of complex mixtures of organic compounds.

Based on the mechanism of interaction of the substance being separated (eluate) with the stationary phase, adsorption, partition, ion exchange, 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 being separated with an adsorbent, such as aluminum oxide or silica gel, which has active polar centers on the surface. The solvent (eluent) is a non-polar liquid. The mechanism of sorption consists of a specific interaction between the polar surface of the sorbent and polar (or capable of polarization) sections of the molecules of the analyzed component (Fig. 1).

Rice. 1. Adsorption liquid chromatography.

Partition chromatography. In the distribution 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 the sorbent (most often silica gel). Substituted alkylchlorosilanes containing polar groups such as nitrile, amino group, etc. are used as silica gel surface modifiers (grafted phases) (Fig. 2). The use of grafted 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 grafted phase (normal-phase option).

Reversed phase liquid chromatography is based on the distribution of mixture components between a polar eluent and non-polar groups (long alkyl chains) grafted onto the surface of the sorbent (Fig. 3).

Rice. 3. Partition chromatography with grafted phase (reversed phase option).

A less widely used variant of supported phase liquid chromatography is where a liquid stationary phase is deposited on 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 the sorbent (synthetic resin), which selectively form complexes (conjugates) with proteins.

Ion exchange, ion pair, and 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 retention time of the mixture component (Fig. 4).

Retention time tR is the time elapsed from the moment the sample is introduced into the column until the maximum of the corresponding peak is released. Multiplying the retention time by the volumetric velocity of the eluent F, we obtain the retention volume VR:

Corrected retention time is the time elapsed from the appearance of the maximum peak of the unsorbed component to the peak of the corresponding compound:

tR" = tR - t0 ;

The normalized or corrected retention volume is the retention volume corrected for the column dead volume V0, i.e., the retention volume of the unsorbed component:

VR" = VR - V0;

A characteristic of retention is also the capacity coefficient k", defined as the ratio of the mass of a substance in the stationary phase to the mass of a substance in the mobile phase: k" = mn / mp;

The k" value can be easily determined 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 theoretical plates (HETP) and inversely proportional to their number (N), is higher, the narrower the peak of the substance released 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- retention 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 height equivalent to the theoretical plate (HETT) and the reduced height (RHETT):

HETT = L/N PVET = HETT/d c

These characteristics make it possible to compare the efficiency of different types of columns, evaluate the quality of the sorbent and the quality of filling the columns.

The selectivity for separating 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 are related by the following equation for resolution:

;

The factors determining the selectivity of separation are:

1) chemical nature of the sorbent;

2) composition of the solvent and its modifiers;

3) chemical structure and properties of the components of the mixture being separated;

4) column temperature

1.1 Equipment for liquid chromatography

In modern liquid chromatography, instruments of varying degrees of complexity are used - from the simplest systems to high-class chromatographs equipped with various additional devices.

In Fig. 4. A block diagram of a liquid chromatograph is presented, 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 flow of solvent. Its design is determined primarily by the operating pressure in the system. To operate 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 to fill with eluent, and the second is the greater complexity of the design and, as a consequence, 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 constant pressure and flow rate, their use is limited to preparative tasks.

The injector (3) ensures that a sample of a mixture of the components being separated is introduced into the column with fairly high reproducibility. Simple "stop-flow" sample injection systems require stopping the pump and are therefore less convenient than the loop pipettes developed by Reodyne.

The HPLC columns (4) are thick-walled stainless steel tubes that can withstand high pressures. The density and uniformity of packing of the column with sorbent plays an important role. Thick-walled glass columns are successfully used for low-pressure liquid chromatography. Constant temperature is ensured by a thermostat (5).

Detectors (6) for liquid chromatography have a flow cell in which a continuous measurement of some property of the flowing eluent occurs. The most popular types of general purpose detectors are refractometers, which measure 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 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 in this case is impossible.

HPLC columns, which are most often used in the analysis of environmental pollutants, are 25 cm long and have an internal diameter of 4.6 mm, and are packed with 5-10 µm spherical silica gel particles grafted with octadecyl groups. In recent years, columns with smaller internal diameters filled with smaller particles have become available. The use of such columns leads to a reduction in solvent consumption and analysis time, an increase in sensitivity and separation efficiency, and also simplifies the problem of connecting columns to spectral detectors. Columns with an internal diameter of 3.1 mm are equipped with a safety cartridge (pre-column) to increase service life and improve analytical reproducibility.

The detectors used in modern HPLC instruments are usually a UV diode array detector, a fluorescent detector, and an electrochemical detector.

It should be borne in mind that in practical work, separation often occurs not through one, but through several mechanisms simultaneously. Thus, exclusion separation can be complicated by adsorption effects, adsorption separation by distribution effects, and vice versa. Moreover, the greater the difference between the substances in the sample in terms of the degree of ionization, basicity or acidity, molecular weight, polarizability and other parameters, the greater the likelihood of a different separation mechanism for such substances.

In practice, the most widespread is “reverse phase” (distribution) chromatography, in which the stationary phase is not polar, but the mobile phase is polar (i.e., the reverse of “direct phase” chromatography).

In most laboratories around the world, a group of 16 priority PAHs are analyzed by HPLC or CMS.

CHAPTER 2. ESSENCE OF HPLC

In HPLC, pure solvents or mixtures thereof are usually used as mobile phases.

To create a stream of pure solvent (or mixtures of solvents), called an eluent in liquid chromatography, pumps included in the hydraulic system of the chromatograph are used.

Insufficient mechanical strength, which does not allow packaging and use at high pressures characteristic of HPLC;

silica gel, compared to aluminum oxide, has a wider range of porosity, surface area and pore diameter; The significantly greater catalytic activity of aluminum oxide leads to distortion of analysis results due to the decomposition of sample components or their irreversible chemisorption.

Detectors for HPLC

Detectors:

Fluorescence detector. The great popularity of fluorescent detectors is due to their very high selectivity and sensitivity, and the fact that many environmental pollutants fluoresce (eg 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.

The continuously detected signal is recorded by a recorder. A chromatogram is a sequence of detector signals recorded on a recorder tape, generated when individual components of a mixture leave the column. If the mixture is separated, individual peaks are visible on the external chromatogram. The position of the peak in the chromatogram is used for the purpose of identifying the substance, the height or area of the peak - for the purpose of quantitative determination.

2.1 Application

HPLC is most widely used in the following areas of chemical analysis (objects of analysis where HPLC has virtually no competition are highlighted):

· Food quality control - tonics and flavoring additives, aldehydes, ketones, vitamins, sugars, dyes, preservatives, hormonal drugs, antibiotics, triazine, carbamate and other pesticides, mycotoxins, nitrosamines, polycyclic aromatic hydrocarbons, etc.

· Environmental protection - phenols, organic nitro compounds, mono- and polycyclic aromatic hydrocarbons, a number of pesticides, main anions and cations.

· Forensics - drugs, organic explosives and dyes, potent pharmaceuticals.

· Pharmaceutical industry - steroid hormones, almost 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 diagnosing diseases, determining the rate of elimination 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 fertilizers applied, 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 (almost all soluble compounds in the form of ions and complex compounds).

· control of the quality and safety of food, alcoholic and non-alcoholic drinks, 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 explosive substances;

· determination of the presence of harmful substances (polycyclic and other aromatic hydrocarbons, phenols, pesticides, organic dyes, ions of heavy, alkali and alkaline earth metals) in liquid effluents, air emissions and solid waste from enterprises and in living organisms;

· monitoring of processes of organic synthesis, oil and coal refining, biochemical and microbiological production;

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 microquantities of ultrapure substances.

CHAPTER 3. EXAMPLES OF USING HPLC IN THE ANALYSIS OF ENVIRONMENTAL OBJECTS

HPLC is 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) in natural objects have been established. The determination of PAHs at the MPC level and below is one of the most complex analytical problems, and high-tech analytical methods (GC-MS, GC, HPLC) are used to solve them. When choosing a method for monitoring, to the main characteristics considered - sensitivity and selectivity, speed and efficiency are added, because monitoring involves serial analysis. The HPLC option on short, small-diameter columns largely meets these requirements. Using this method, the authors developed and certified methods for monitoring benzo[a]pyrene in three natural environments: aerosol, snow cover and surface water. The methods are characterized by: simple standardized sample preparation, including extraction of PAHs with organic solvents and concentration of the extract, direct introduction of the concentrated extract into a chromatographic column, use of multi-wavelength 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 benzo[a]pyrene in aerosol in the concentration range from 0.3 to 450 ng/m3, in surface water in the concentration range from 10 to 1000 ng/l, in snow cover in the surface density range from 0.5 up to 50 μg/m2. For the case of simultaneous determination of priority PAHs (up to 12 compounds) and registration of inhomogeneous peaks of analytes, repeated separation of the extract was proposed by changing the selectivity of the mobile phase, detection wavelength and column temperature, taking into account the individual properties of the PAH being determined.

1 . Ambient air quality. Mass concentration of benzo[a]pyrene. Methodology for performing measurements using the HPLC method. Certificate of certification MVI No. 01-2000.

2 . Quality of surface and treated wastewater. Mass concentration of benzo[a]pyrene. Methodology for performing measurements using the HPLC method. Certificate of certification MVI No. 01-2001.

3 . Quality of snow cover. Mass concentration of benzo[a]pyrene. Methodology for performing measurements using the HPLC method. Certificate of certification MVI No. 02-2001.

Removal of aniline from aqueous solutions using waste from aluminothermic reduction of mill 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 sedimentation.

One of the best methods for treating wastewater from organic pollutants is the use of inorganic and organic adsorbents that can be regenerated (aluminosilicates, modified clays, wood, fibers, etc.) and incapable of regeneration (activated carbon, macroporous polymeric materials, etc. ).

Regenerable adsorbents can remove organic substances of different polarities from water. The search for effective adsorbents is an urgent task.

This report presents the results of a study in the field of using rolled copper scale from 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 the sorbents we studied, the mobile phase speed was 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 were varied. The mixture was thoroughly shaken for 6 hours. Then the sample was left to settle. Adsorption is completed almost within 48 hours. The amount of precipitated aniline is determined by UV spectrophotometric as well as refractometric analysis.

First, the adsorption properties of OPMOErKZ were studied when removing aniline from a solution in carbon tetrachloride. It turned out that aniline absorbs sorbent 3 best (table).

Measurements were also carried out for aqueous solutions of aniline in concentrations of 0.01-0.0001 mol/l. The table shows data for a 0.01 M solution.

Absorption of aniline by various sorbents from a 0.01 M aqueous solution of aniline at 20°C

It was previously established that adsorption within the specified concentration ranges increases and linearly depends on the refractive index. The amount of aniline was determined from the graphical relationship “refractive index - molar concentration” and corrected by data from both liquid chromatography and UV spectral analysis.

The most active sorbent for aqueous solutions is sorbent 3. 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 by using either capillary columns in gas chromatography or high-performance columns used in HPLC. It must be remembered that a column that well separates calibration mixtures of sixteen PAHs does not guarantee that they will also be well separated from the background of accompanying organic compounds in the samples under study.

In order to simplify the analysis, as well as to achieve high quality results, most analytical procedures contain a stage of preliminary isolation (separation) of PAHs from other groups of associated compounds in samples. Most often, low-pressure liquid chromatography methods are used for these purposes in a liquid-solid or liquid-liquid system using adsorption mechanisms, for example using silica gel or alumina, sometimes mixed mechanisms are used, for example adsorption and exclusion using Sephadex.

The use of preliminary purification of samples allows one 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 resins.

There are mainly two types of detectors used in high-performance liquid chromatography (HPLC): a fluorimetric detector or a photodiode array spectrophotometric detector. The detection limit of PAHs in fluorimetric detection is very low, making this method particularly suitable for the determination of trace amounts of polyaromatic compounds. However, classical fluorimetric detectors provide practically 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 are not yet widely used in routine measurement practice. A spectrophotometric detector with a photodiode array (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 selecting analytical techniques for the separation, identification and quantitative analysis of these PAHs, the following conditions must be taken into account:

The level of determined contents in the test samples;

Number of related substances;

The analytical procedure used (measurement technique);

Capabilities of serial equipment.

Development of a method for determining alkaline earth elements and magnesium using 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. The development of high-pressure high-performance liquid chromatography 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 there are quite a lot of requirements for them. Due to the lack of commercially available highly effective cation exchangers, a dynamically modified reverse phase was used, for which a modifier was synthesized: N-hexadecyl-N-decanoyl-paraminobenoylsulfonic acid ethyl-diisopropylammonium (DHDAS), 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 optical density units (°E) and reached a plateau. The calculated ion exchange capacity is 15.65 µmol. Since cations of alkaline earth elements and magnesium do not absorb in the UV region of the spectrum, indirect UV detection was used using a synthesized UV-absorbing eluent 1,4-dipyridiniumbutane bromide (DPB bromide). Since halogen ions destroy the steel parts of the column, the bromide ion of 1,4-dipyridiniumbutane was replaced with acetate ion. When washing the column with the 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 “folding scale” mode; The polarity of the recorder was reversed. The separation of all studied cations was achieved with the addition of a complexing additive - oxalic acid. The detection limits for 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 was 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 existence of metals in nature is required. Difficulties in analyzing compounds of one of the most toxic metals - cadmium - are due to the fact that it forms fragile complexes, and when trying to isolate them, natural equilibria are distorted. In this work, cadmium compounds in soil and plants were studied using a technique based on chromatographic separation of extracts followed by identification of components by chemical analysis methods. This approach made it possible not only to identify the chemical forms of cadmium, but also to trace their transformations in environmental objects.

The OH groups of carbohydrates and polyphenols (including flavonoids), C=O, phosphates, NH 2 , NO 2 , and 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 using UV spectroscopy and HPLC.

To isolate cadmium compounds, extraction with specially selected (not forming complexes with Cd) solvents was used. This makes it possible to separate cadmium from all heavy metals, except 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 stability of Cd and Zn complexes at pH 6-8 was used. The isolated Cd compounds were identified by HPLC using pH changes during the elution process. An analysis of cadmium compounds with components of soils and plant tissues was carried out, and substances produced by plants in response to an increase in the supply 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 vegetables.

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 separation on conventional columns and ultra-fast and efficient separation on columns with a sorbent particle size of less than 2 microns at ultra-high pressures (more than 1000 atm.).

The system includes a quadrant gradient pump capable of generating pressures in excess of 1000 atm and with a retention volume of only 65 µL, enabling high-speed chromatographic separations. Autosampler ACCELA Capable of operating at a sample injection cycle of 30 seconds and providing the highest injection reproducibility. Diode array detector Accela PDA with a minimized flow cell volume (2 µl) optimized for high speed chromatography, uses patented LightPipe technology and maintains the symmetrical peak shapes that come with a flawless chromatography system and columns.

The system interfaces seamlessly 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 any application

SERIES TSP

The modular principle of constructing HPLC instruments allows the customer to flexibly assemble equipment to solve any analytical problems, and if they change, quickly and economically modify it. A wide selection of modules includes pumps - from isocratic to four-component gradient, from microcolumn to semi-preparative, all available detectors, sample introduction systems - from manual injectors to autosamplers with the possibility of any manipulation with samples, powerful software for processing measurement results and managing all system modules. All modules are certified by 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-diagnosis system, allow you to create and save task methods. parameters. They meet the criteria of “Exemplary Laboratory Practice” (GLP) and are included in the Register of Measuring Instruments of the Russian Federation. Measurement reports 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. Whatever the High Performance Liquid Chromatography (HPLC) application - pharmaceuticals, biotechnology, environmental analysis, clinical analysis, food and beverage analysis, petrochemical and chemical analysis - this instrument is always optimally configured to suit to the highest requirements.

Both research and high-throughput 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 optional 1 or 5 cm optical path length)

Working with different columns

Highest precision quantitative analysis

Ability to automatically work with different sample volumes

RMS error for retention times less than 0.3%

Minimum working area occupied by the system

Highest reliability and stability of parameters.

Surveyor LC Pump- HPLC pump with the best retention time reproducibility of any four-component gradient pump available in the world. Integrated four-channel vacuum degasser and pulsation damper provide excellent baseline stability for maximum sensitivity and quantitation accuracy.

The autosampler provides the highest productivity 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. The new technology ensures sample injection with virtually no loss; almost 5 µl of sample is injected with an autosampler from a total sample volume of 5 µl.

SURVEYOR

UV/Vis detector and PDA (Diode Array Detector)

Surveyor UV/Vis- The variable wavelength ultraviolet and visible light detector is a combination of cost-effectiveness 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 semipreparative and preparative.

Surveyor PDA The detector is the most sensitive of all HPLC detectors using a diode array. 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 R.I. refractometric detector with a thermostated cuvette of minimal volume with full electronic control from a computer.

Surveyor FL fluorimetric scanning detector with the highest sensitivity and detection capabilities for fluorescence, chemiluminescence and phosphorescence.

A wide selection of autosamplers allows you to work with both conventional vials and 96-position plates, widely used in biochemistry and clinical practice. Working with them is facilitated by the use of similar plates for sample preparation using solid-phase extraction.

400 Electric drive, Valco loop (20 µl - standard) with partial filling capability.

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 operate in full, partial filling and microliter sample injection modes. Wide selection of carousels (standard - 96 samples).

Tablet autosampler for working with 96- and 384-position plates. Injection of sample into the loop under pressure, the ability to inject samples less than 1 µl. Possibility of installing a tablet feeder. HPLC

Major manufacturers of HPLC equipment

· Waters - ultra-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: book..1 - M.: Chemistry, 1990, -480 p.

1. Pilipenko A.T., Pyatnitsky I.V. Analytical chemistry. In two books: book..2 - M.: Chemistry, 1990, -480 p.

2. Vasilyev V.P. Analytical chemistry. In 2 hours. Part 2. Physico-chemical methods of analysis: Textbook. for Khimko - technol. specialist. universities – M.: Higher. school, 1989. – 384 p.

3. Hydrochemical materials. Volume 100. Methods and technical means of operational monitoring of surface water quality. L.: Gidrometeo-izdat, 1991. – 200 p.

4. Lurie Yu.Yu. Analytical chemistry of industrial wastewater / Yu.Yu. Lurie; M.: KhimiyaYU, 1984. - 448 p.

5. Ewing G. Instrumental methods of chemical analysis / Transl. from English M.: Mir, 1989. – 348 p.

6. Gorelik D.O., Konopelko L.A., Pankov E.D. Environmental monitoring. In 2 volumes. 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. - P. 81.

11. Gorshkov A.G., Marinaite I.I. HPLC is 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 from aluminothermic reduction of rolled copper scale

13. L.A. Turkina, G.N. Koroleva Development of a method for determining alkaline earth elements and magnesium using 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 of Hygiene named after. F.F.

Erisman; Moscow Agricultural Academy named after. K.A.

Timiryazev; with the participation of the Department of State Sanitary and Epidemiological Surveillance of the Russian Ministry of Health. 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, academician. RAMS G.G. Onishchenko June 24, 2003

5. Introduced for the first time.

1. Introductory part

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.m. dm. Stable at room temperature for at least 2 years, at 50 -C for at least 3 months.

Brief toxicological characteristics: 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/m3. dm (4 hours).

Hygienic standards. Maximum concentration limit in water is 0.02 mg/m3. dm.

Area of application of the drug. Clomazone is a selective herbicide used to control cereals and dicotyledonous weeds in soybean and rice crops with pre-emergence or pre-sowing application.

2. Method for determining clomazone in water

chromatographic methods

2.1. Basic provisions

2.1.1. 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 absolute calibration method.

2.1.2. Selectivity of the method

Under the proposed conditions, the method is specific in the presence of global environmental pollutants: chlorinated cycloparaffins (HCCH 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 characteristics of the method (P = 0.95)

Reagents, solutions and materials

Clomazone containing d.v. 99.8%

(FMS, USA)

Nitrogen, very high GOST 9293-79

Aqueous 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, reagent grade GOST 4203-77

Hydrochloric acid (hydrochloric acid), reagent grade GOST 3118-77

Methyl alcohol, reagent grade GOST

Sodium hydroxide, chemically pure, 25% aqueous solution GOST 4323-77

Sodium sulfate anhydrous, reagent grade GOST 1277-81

Silver nitrate, reagent grade GOST 1277-81

2-Phenoxymethanol, part 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)

Plates "Kieselgel 60 F-254" (Germany)

Records "Silufol" Czech Republic

Paper filters "white tape", deashed and pre-washed with hexane TU 6-09-2678-77

2.3. Devices, equipment, dishes

Liquid chromatograph Milichrome

with ultraviolet detector

Steel chromatographic column,

length 64 mm, internal diameter 2 mm,

filled with Silasorb 600, grain size 5 microns

Gas chromatograph series "Color" or

similar, equipped with a constant detector

recombination rate (RPR) with a limit

detection for lindane 4 x 10 g/cc. cm

Glass chromatographic column, length

1 or 2 m, internal diameter 2 - 3 mm

Microsyringe type MSh-10, capacity 10 µl TU 5E2-833-024

Shaking apparatus type AVU-6s TU 64-1-2851-78

Water bath TU 64-1-2850-76

Analytical balances 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

Glass spray bottles 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

Separating funnels GOST 3613-75

Volumetric flasks, capacity 100 ml GOST 1770-74

Measuring cylinders, capacity 10, 50 ml GOST 1770-74E

Pear-shaped flasks with ground section,

with a capacity of 100 ml GOST 10394-72

Conical flasks, capacity 100 ml GOST 22524-77

Centrifuge tubes, measuring GOST 25336-82E

Pipettes, capacity 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

Selection, storage and preparation of samples are carried out in accordance with

"Unified rules for sampling agricultural products, food products and environmental objects to determine trace amounts of pesticides", approved by N 2051-79 of 08.21.79

Selected samples can be stored in the refrigerator for no more than 5 days. Before analysis, the water (if suspended matter is present) is filtered through a loose paper filter.

2.5. Preparing for determination

2.5.1. HPLC method

2.5.1.1. Preparation of the mobile phase for HPLC

Using a pipette, place 5 ml of isopopanol and 5 ml of methanol into a 100 ml volumetric flask, add hexane to the mark, mix, and filter.

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 flow 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 without connecting to the detector, and stabilized in a nitrogen stream at a temperature of 250 -C for 10 - 12 o'clock

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 to 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 flask.

2.5.3.2. Preparation of mobile phase for TLC

Add 20 ml of acetone to a 100 ml volumetric flask, add hexane to the mark and mix. The mixture is poured into the chromatographic chamber in a layer of no more than 6 - 8 mm in 30 minutes. Before starting chromatography.

2.5.4. Preparation of standard solutions

A stock standard solution of clomazone containing 100 µg/ml is prepared by dissolving 0.010 g of the drug containing 99.8% active ingredient 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 the stock standard solution of clomazone by appropriate serial dilution 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 graph A (measurement according to clause 2.7.1, HPLC)

To construct 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 graph B (measurement according to clause 2.7.2, GLC)

To construct a calibration curve, 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 chromatographic peak height for each concentration. Construct a calibration graph (A or B) of the dependence of the height of the chromatographic peak in mm on the concentration of clomazone in the 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 100 ml pear-shaped flask, passing it through a layer of anhydrous sodium sulfate placed in a conical funnel on a folded paper filter. 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 high-purity nitrogen. 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, internal diameter 2 mm,

filled with Silasorb 600, grain size 5 microns.

Column temperature: room temperature.

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.

Injected sample volume: 5 µl.

Clomazone release time: about 6 minutes.

Linear detection range: 20 - 200 ng.

Samples producing peaks larger than the 40 µg/mL standard solution are diluted with HPLC-grade mobile phase.

Gas chromatograph "Tsvet-570" with a detector of constant ion recombination rate.

Glass column 1 m long, internal diameter 3 mm, filled with Chromaton N-AW-DMCS with 5% SE-30 (0.16 - 0.20 mm).

The operating scale of the electrometer is 64 x 10 10 Ohm.

The speed of the recorder tape is 200 mm/h.

Column thermostat temperature - 190 -C

detector - 300 -С

evaporator - 220 -С

Carrier gas (nitrogen) speed - 60 ml/min.

The volume of the injected sample is 5 µl.

Clomazone release time is 2.5 minutes.

Linear detection range: 2 - 50 ng.

Samples producing peaks larger than the 10 μg/mL standard solution are diluted with hexane.

To increase the accuracy of the identification of clomazone in the presence of gamma-HCH in the sample, which has a similar retention time, clomazone is removed from the sample by treatment with concentrated sulfuric acid. Repeated analysis of the sample makes it possible to determine the contribution of clomazone to the primary chromatographic signal.

Hexane solution in a flask obtained according to paragraph 2.6 quantitatively

(or an aliquot of it) is applied to chromatographic plates “Silufol”, “Kieselgel 60F-254” or “HPTLC plates”. Standard solutions are applied nearby in a volume corresponding to the clomazone content of 1, 2, 5 and 10 mcg. The plate is placed in a chromatography chamber containing a mixture of n-hexane-acetone (4:1, by volume). After the chromatogram has developed, the plate is removed from the chamber, placed under traction 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 “Silufol” plates, “HPTLC plates” and “Kieselgel 60F-254” appears in the form of gray-brown spots with an Rf value of 0.35, 0.85 and 0.43, respectively. To determine clomazone by TLC, you can use “Alugram” and “Poligram” plates (made in 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, and electric heating devices.

4. Measurement error control

Operational control of measurement error and reproducibility 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. (Federal Research Center 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 Academy of Medical Sciences of Ukraine, Kyiv).

(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 chromatography column filled with inaccessible

visual phase (sorbent). HPLC columns are characterized by high hydraulic pressure at the column inlet, so HPLC is sometimes called

called "high pressure liquid chromatography".

Depending on the mechanism of separation of substances, the following are distinguished:

current HPLC options: adsorption, distribution, ion exchange,

exclusive, chiral, etc.

In adsorption chromatography, the separation of substances occurs due to their different abilities to be adsorbed and desorbed with

the surface of an adsorbent with a developed surface, for example, silica gel.

In distribution HPLC, separation occurs due to differences in the distribution coefficients of the substances being separated between the stationary

(usually chemically grafted onto the surface of a stationary carrier) and

mobile phases.

By polarity, PF and NP HPLC are divided into normal-phase and ob-

extended-phase.

Normal-phase is a variant of chromatography in which

use a polar sorbent (for example, silica gel or silica gel with

twisted NH2 - or CN groups) and non-polar PF (for example, hexane with di-

personal supplements). In the reversed-phase version of chromatography,

use non-polar chemically modified sorbents (for example,

non-polar alkyl radical C18) and polar mobile phases (e.g.

methanol, acetonitrile).

In ion exchange chromatography, molecules of substances in a mixture, dissociate

in solution into cations and anions are separated when moving through

sorbent (cation exchanger or anion exchanger) due to their different rates of exchange with ion-

mi groups of sorbent.

In exclusion (sieve, gel-permeating, gel filtration)

chromatography, molecules of substances are separated by size due to their different ability to penetrate the pores of the stationary phase. At the same time, the first of the co-

The columns yield the largest molecules (with the highest molecular weight) capable of penetrating into the minimum number of pores of the stationary phase,

and the last to emerge are substances with small molecular sizes.

Often separation occurs not through one, but through several mechanisms simultaneously.

The HPLC method can be used to control the quality of any non-

zooform analytes. To carry out the analysis, appropriate instruments are used - liquid chromatographs.

The composition of a liquid chromatograph usually includes the following basic elements:

Nodes:

– PF preparation unit, including a container with a mobile phase (or capacitive

ties with individual solvents included in the mobile phase

3) and the PF degassing system;

– pumping system;

– mobile phase mixer (if necessary);

– sample introduction system (injector);

– chromatographic column (can be installed in a thermostat);

– detector;

– data collection and processing system.

Pumping system

Pumps supply PF to the column at a given constant speed. The composition of the mobile phase can be constant or variable.

during the analysis. In the first case, the process is called isocratic,

and in the second - gradient. They are sometimes installed in front of the pumping system

filters with a pore diameter of 0.45 microns for filtration of the mobile phase. Modern

A liquid chromatograph pump system consists of one or more computer-controlled pumps. This allows you to change the

becoming PF according to a certain program with gradient elution. Sme-

The mixture of PF components in the mixer can occur both at low pressure

pressure (before the pumps) and at high pressure (after the pumps). The mixer can be used for the preparation of PF and isocratic elution,

however, a more accurate ratio of components is achieved by pre-

thorough mixing of PF components for an isocratic process. Pumps for analytical HPLC 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 pressure at the column inlet of up to 50 MPa. It is advisable, however, that this value does not exceed

shal 20 MPa. Pressure pulsations are minimized by special dampers

ferrous systems included in the pump design. Working parts on-

The pumps are made from corrosion-resistant materials, which allows the use of aggressive components in the PF composition.

Faucets

By design, mixers can be static or dynamic

mic.

In the mixer, a single mobile phase is formed from the

efficient solvents supplied by pumps if the required mixture has not been prepared in advance. Mixing of solvents usually occurs spontaneously, but sometimes systems with forced mixing are used.

sewing.

Injectors

Injectors can be universal for introducing samples from

1 µl to 2 ml or discrete for introducing a sample of only a certain volume

ema. Both types of injectors can be automatic ("autoinjectors" or "autosamplers"). The injector for introducing the sample (solution) is located

directly in front of the chromatography column. The design of the injector allows you to change the direction of the PF flow and pre-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 injector design allows for loop replacement. To introduce the test solution into a non-automatic

Tomatic injector uses a manual microsyringe with a volume of significantly

significantly exceeding the volume of the loop. Excess of injected solution, not

located in the loop is reset, and an exact and always equal volume of sample is injected into the column. Manually incompletely filling the loop reduces the accuracy

accuracy and reproducibility of dosing and, therefore, impairs the accuracy

accuracy and reproducibility of chromatographic analysis.

Chromatographic column

Chromatographic columns are usually tubes made of stainless steel, glass or plastic, filled with a sorbent and closed.

lined on both sides with filters with a pore diameter of 2–5 microns. Analytical length

The thickness of the column, depending on the chromatographic separation mechanism, can be in the range from 5 to 60 cm or more (usually it is

10–25 cm), internal diameter – from 2 to 10 mm (usually 4.6 mm). Columns with an internal diameter of less than 2 mm are used in microcolumn chromium

tography. Capillary columns with an internal diameter of

rum about 0.3-0.7 mm. Columns for preparative chromatography have an internal diameter of up to 50 mm or more.

Short tubes can be installed in front of the analytical column.

columns (pre-columns) performing various auxiliary functions

(more often – protection of the analytical column). Usually the analysis is carried out with

at normal temperature, however, to increase the efficiency of separation and con-

To shorten the duration of analysis, thermostats can be used.

testing of columns at temperatures not higher than 60 C. At higher temperatures, destruction of the sorbent and changes in the composition of the PF are possible.

Stationary phase (sorbent)

The following are usually used as sorbents:

1. Silica gel, aluminum oxide, porous graphite are used in normal

small-phase chromatography. The retention mechanism in this case is

tea - usually adsorption;

2. Resins or polymers with acidic or basic groups. Area of application: 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 distribution between movable

new and stationary phases;

5. Chemically modified chiral sorbents, for example those produced

aqueous celluloses and amyloses, proteins and peptides, cyclodextrins,

used for the separation of enantiomers (chiral chromatography

Sorbents with grafted phases can have varying degrees of chemical

ical modification. Sorbent particles can be spherical or non-spherical

correct shape and varied porosity.

The most commonly used grafted phases are:

– octyl groups(octylsilane or C8 sorbent);

– octadecyl groups(sorbent octadecylsilane

(ODS) or C18);

– phenyl groups(phenylsilane sorbent);

– cyanopropyl groups(CN sorbent);

– aminopropyl groups(NH2 sorbent);

– diol groups (sorbent diol).

Most often, analysis is performed on non-polar grafted 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 grafted phases (“CN”, “NH2”, “diol”) are used in combination with non-polar solutions.

Sorbents with grafted phases are chemically stable at pH values from 2.0 to 8.0, unless otherwise specifically stated by the manufacturer.

Sorbent particles can have spherical or irregular shapes and varied 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.

High separation efficiency is ensured by the high surface area of sorbent particles (which is a consequence of their microscopic

ical size and presence of pores), as well as the uniformity of the sorbent composition 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 enters 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

logical or physicochemical parameter of PF as a function of time.

The most common are spectrophotometric de-

tectors (including diode-matrix) that record changes in optical

density in the ultraviolet, visible and often in the near infrared

n areas of the spectrum from 190 to 800 or 900 nm. The chromatogram in this case

tea represents 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. Multi-wave detectors are also used, allowing

Perform detection at several wavelengths simultaneously.

Using a diode array detector, you can not only detect several wavelengths at once, but also almost instantly

It is possible to obtain the optical spectrum of the PF directly (without scanning) at any time, which greatly simplifies the qualitative analysis of the separated components.

ponents.

The sensitivity of fluorescence detectors is approximately 1000 times higher than the sensitivity of spectrophotometric ones. In this case, either its own fluorescence or the fluorescence of the corresponding derivatives is used if the substance being determined itself does not fluoresce. Modern

variable fluorescent detectors allow not only to obtain chromato-

grams, but also to record the excitation and fluorescence spectra of the analytical

zirovable connections.

To analyze samples that do not absorb in the UV and visible regions of the spectrum (for example, carbohydrates), refractometric detectors are used

(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).

Electrochemical detectors are also used (conductometric

ski, amperometric, etc.), mass spectrometric and Fourier-IR

detectors, light scattering detectors, radioactivity detectors and some other

Mobile phase

IN A variety of solvents can be used as PF - both individual and mixtures thereof.

IN normal-phase chromatography usually uses liquid carbon

hydrochlorides (hexane, cyclohexane, heptane) and other relatively non-polar

solvents with small additions of polar organic compounds,

which regulate the elution 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 optical

separation systems often use aqueous solutions with a certain

pH changes, in particular buffer solutions. Inorganic additives are used

chemical and organic acids, bases and salts and other compounds (for example,

example, chiral modifiers for separating enantiomers into achiral-

nom sorbent).

The pH value must be monitored separately for the aqueous component, and not for its mixture with an organic solvent.

PF can consist of one solvent, often two, if necessary

range - three or more. The composition of the PF is indicated as the volume ratio of its constituent solvents. In some cases, the mass may be indicated

owl ratio, which should be specially stipulated.

When using a UV spectrophotometric detector, the PF should not have pronounced absorption at the wavelength selected for detection. Transparency limit or optical density when determined

The specific wavelength of a solvent from a particular manufacturer is often indicated

is on the packaging.

The chromatographic analysis is greatly influenced by the degree of purity of the PF, therefore it is preferable to use solvents produced

specially designed for liquid chromatography (including water).

PF and analyzed solutions should not contain undissolved

of particles and gas bubbles. Water obtained in laboratory conditions

aqueous solutions, organic solutions pre-mixed with water

The media, as well as the analyzed solutions, must be subjected to fine filtration and degassing. Filtration is usually used for these purposes.

under vacuum through a membrane filter with a pore size of 0.45 μm, inert to a given solvent or solution.

Data collection and processing system

A modern data processing system is a con- nected

a personal computer connected to a chromatograph with installed software

software that allows you to register and process chro-

matogram, as well as control the operation of the chromatograph and monitor the main

parameters of the chromatographic system.

List of chromatographic conditions to be specified

The private pharmacopoeial monograph must indicate the dimensions of the co-

columns, type of sorbent indicating particle size, column temperature (if thermostatting is necessary), volume of injected sample (loop volume),

becoming PF and the method of its preparation, PF supply 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 the analysis of both organic

both (heterocyclic bases, amino acids, proteins, etc.), and non-or-

ganic (various cations and anions) compounds. Composite separation

nents of the analyzed mixture in ion exchange chromatography is based on the reversible interaction of ions of the analyzed substances with ionic groups

memory of the sorbent. Anion exchangers or cation exchangers are used as sorbents.

You. These sorbents are mainly either polymer ion-

exchange resins (usually copolymers of styrene and divinylbenzene with

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

cation removal – modified silica gels.

Aqueous solutions of acids, bases and salts are used as PFs in ion exchange chromatography. Usually buffers are used

solutions that allow you to maintain certain pH values. It is also possible to use small organic additives that mix with water.

ski solvents - acetonitrile, methanol, ethanol, tetrahydrofuran.

Ion chromatography- a variant of ion exchange chromatography, in

in which, to determine the concentration of ions of the analyte, we use

uses 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 should be low.

There are two main variants of ion chromatography.

The first of them is based on suppressing the electrical conductivity of electrolytic

PF using a second ion exchange column located between the

lytic column and detector. Neutralization occurs in this column

tion of the PF and the analyzed compounds enter the detector cell in deionization

distilled water. The detected ions are the only ions

ensuring conductivity of the PF. The disadvantage of the suppression column is the need for its regeneration after fairly short periods of time.

me. The suppression column can be replaced continuously

a common membrane suppressor in which the composition of the membrane is continuously

is renewed by a flow of regenerating solution moving in the direction

opposite to the direction of PF flow.

The second version of ion chromatography is single-column ion chromatography.

matography. In this embodiment, a PF with a very low electrical conductivity is used.

water content. Weak organic compounds are widely used as electrolytes.

Chinese acids - benzoic, salicylic or isophthalic.

SECURE HPLC

Size exclusion chromatography (gel chromatography) is a special version of HPLC based on the separation of molecules by 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 microns.

The advantages of porous glasses and silica gel are the rapid diffusion of PF and molecules of the analyzed substance into the pores, stability in various conditions (even at high temperatures). Polymer sorbene-

you are copolymers of styrene and divinylbenzene (this is 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 whose size is larger than the average pore diameter do not penetrate the sorbent at all and elute together with the mobile phase.

zoy first. Molecules with a diameter significantly smaller than the pore size of the sorbent

benta freely penetrate into it, remain in the stationary phase for the longest time and are the last to elute. Medium-sized molecules penetrate into the pores of the sorbent depending on their size and partly 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

the diffusion of 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

research of water-soluble compounds on hydrophilic sorbents. Mobile phases are aqueous buffer solutions with a given pH value.

In gel permeation chromatography, hydrophobic sorbs are used.

bents and non-polar organic solvents (toluene, dichloromethane, te-

ragidofuran). This method is used for the analysis of compounds that are poorly soluble

rims in the water.

Detectors. Differential refractometric detectors, as well as spectrophotometric detectors (including in the IR spectral region) are used as detectors in size exclusion chromatography.

Viscometer and flow laser detectors are also used.

These detectors, in combination with a refractometer or other concentration

detector make it possible to continuously determine the molecular mass

limer in PF.

ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY

Ultraperformance liquid chromatography is a variant of liquid chromatography that is more efficient

sity compared to classical HPLC.

A feature of ultra-performance liquid chromatography is

The use of sorbents with particle sizes from 1.5 to 2 microns is possible. Dimensions chrome

matographic columns are usually from 50 to 150 mm in length and from 1

up to 4 mm in diameter. The volume of the injected sample can range from 1 to 50 µl.

Chromatographic equipment used in classical va-

riant HPLC, usually specially adapted for this type of chromatogra-

Equipment designed for ultra-performance liquid chromatography can also be used in the classical version of HPLC.

HPLC chromatography. High performance liquid chromatography (HPLC). Liquid adsorption chromatography column

HPLC is a method for monitoring PAHs in environmental objects

11. Gorshkov A.G., Marinaite I.I. HPLC is a method for monitoring PAHs in environmental objects

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