Identification and quantitative determination of pesticides in plant materials by chromatographic methods Ostroukhova Olga Konstantinovna. Thin-layer chromatography of pesticide residues in foods Estimation of hydrophobicity parameters

« Thin-layer chromatography of residual concentrations of pesticides in food products»

INTRODUCTION

Chapter 1. Fundamentals of Planar (Thin Layer) Chromatography

Chapter 2. Status and prospects for the use of modern instrumental methods for the analysis of pesticides

Chapter 3. Guidelines for the determination of organochlorine pesticides in water, food, feed and tobacco products by thin layer chromatography

Chapter 4

Literature

INTRODUCTION

Chemicals (insecticides, herbicides, fungicides) are used to fertilize the soil, control weeds, insects and rodents, and protect crops from mold and fungi. With their help, they increase productivity, increase the shelf life of plants, improve the appearance of fruits, vegetables and grains. Today there is a choice of 5,000 types of pesticides and 700 chemical ingredients. Compared to the beginning of the 1940s, when pesticides were first used, their consumption in agriculture has increased tenfold, and crop losses due to for insects have doubled over the past 50 years. This statistic casts doubt on the "effectiveness" of pesticides. Interestingly, the use of pesticides has led to the development of 650 pest species that are resistant to some of these poisons.
Every day around 3,000 people in the world are poisoned by pesticides. That's over a million poisonings a year from chemicals that pollute the air, soil, water, and food. Separately for Europe, these figures are no less shocking. Only in 2005 did the EU countries begin to try to introduce common standards in assessing the danger of chemicals entering food, and a single label for food. It is known that many pesticides are hazardous to health and have carcinogenic properties, but so far the buyer cannot determine from the label how saturated the purchased product is with these unhealthy substances. In developed countries, the consumer, in principle, has a choice - to buy "organic" (grown without chemicals) products, or conventional. The difference in price is very significant, and the choice of "organic" products is not as great as the usual ones.

The environmental protection organization admits that out of 320 pesticides approved for use in agronomy, at least 66 -
suspected carcinogens. Many of these pesticides are mixed with 1,200 neutral ingredients, whose ingredients are not required to be disclosed by manufacturers, citing "trade secrets". For 800 of them, toxicity levels have not yet been established, they are suspected to be carcinogens. , so it is necessary use methods for identifying pesticides in food.

CHAPTER 1. BASICS OF PLANAR (THIN LAYER) CHROMATOGRAPHY

planar (thin layer) chromatography

Thin-layer (planar) chromatography occupies one of the leading places in the qualitative and semi-quantitative analysis of complex natural, pharmaceutical, biomedical and chemical objects. Among other chromatographic methods, planar chromatography is distinguished by the following advantages and features:

This is the only chromatographic method that allows a complete analysis of an unknown mixture, since the researcher has the opportunity to check if there are any uneluted components left at the start;

Outperforms gas and

high performance liquid chromatography, at least an order of magnitude; uses simpler and cheaper equipment;

It has high selectivity, which is easy to vary by choosing the composition of the mobile phase; unlike HPLC, there are no restrictions on the choice of solvents;

Allows simultaneous separation of several samples; the use of single or multiple elution (under different conditions), as well as the simultaneous separation of the components of the same sample using different eluents;

Resolution optimization possible

chromatographic system when separating a complex mixture only for the components of interest, which saves time;

It is possible to detect compounds with high

sensitivity and selectivity, which can be easily varied by selecting a developing reagent; the obtained separation results are easy to evaluate visually;

You can save chromatograms for later

detection and carry out spectral identification

chromatographic zones after separation in any wavelength range, including IR.

Planar chromatography also has some disadvantages:

Limited separating capacity due to the relatively small length of the separating zone (3-10 cm);

The sensitivity is lower than in the case of HPLC;

Dependence of the results of the analysis on the environment: relative humidity, temperature, as well as the presence of pollutants in the air;

Difficulties in working with highly volatile samples, as well as with substances sensitive to the action of atmospheric oxygen or light.

The classic, most simple and widely used thin layer chromatography technique includes the following main operations:

applying the analyzed sample to the sorbent layer;

separation of the sample components into separate zones in the mobile phase flow;

3) detection of zones on the sorbent layer (often with a reagent that forms colored compounds with separated substances);

4) quantitative evaluation of the obtained separation, including the determination of the retention value and the determination of the content of the substance in the zones on the chromatogram.

The position of the substance zone on the chromatogram is characterized by the R f value, which is equal to the ratio of the distance from the starting line to the center of the substance zone to the distance from the starting line to the front line. The Rf value is a constant value for a given compound in this system and depends on a number of conditions: the elution method, the quality and activity of the sorbent, the layer thickness, the quality of the solvents, the amount of applied substance, the length of the run of the solvents, the position of the starting line, and almost does not depend on temperature. This value is used to identify the components in the mixture.

The quality of separation of mixture components in planar chromatography is affected by a large number of factors: the type of separation chamber; preliminary saturation of the chamber and the sorbent layer with vapors of the mobile phase; starting spot size; distance from the start to the bottom edge of the plate; relative humidity of the air in the laboratory room; average particle diameter and shape; thickness and uniformity of application of the sorbent layer; the presence of microdamages of the layer; type of substance that binds the sorbent; elution rate; solvent volume in the chamber; the presence of impurities in the eluent; convection in the gas phase inside the chamber.

To separate mixtures of substances in a thin layer of a sorbent, adsorption, partition, and ion-exchange chromatography is used, which differ primarily in the nature of the interactions between the dissolved substances and the solid or liquid phases with which they come into contact. In practice, these interactions almost never occur in isolation, and the separation of substances is due to several interactions. When choosing a suitable chromatography option, first of all, attention should be paid to the structure of the substances to be separated. With the help of adsorption and partition chromatography, substances are separated, the structure of which differs in the nature, number and nature of polar and non-polar substituents. When chromatography in a thin layer of a sorbent, adsorption chromatography is most often used, which is simpler to perform, more efficient, and the analysis results are more reproducible.

Sorbents in thin layer chromatography

As sorbents in TLC, materials are used that meet the following requirements: form chemically and physically stable layers; do not form covalent bonds with the separated substances; do not dissolve in the mobile phase or move along with it along the plate; do not contain components that interfere with separation or detection; do not have their own color; do not swell or shrink under the action of the mobile phase.

Glass, aluminum foil, polymer films (polyethylene terephthalate) are used as a substrate for the sorbent. To impart stability to the sorbent layer on the substrate, various binders are used: gypsum (5-10%), silicasol, alkali metal silicates, polyacrylamide, polyacrylic ether, starch. A fluorescent indicator is often added to the adsorbent to detect substances that absorb in the UV region of the spectrum. For this purpose, use: a mixture of zinc and magnesium silicates; a mixture of zinc and cadmium sulfides; tungstates of alkaline earth elements.

Of great importance, especially for separation efficiency, are such characteristics of sorbents as particle diameter, average particle size distribution and pore size. In classical thin layer chromatography, particles with a size of 5 - 20 µm are used to produce plates. High performance thin layer chromatography (HPTLC) requires a sorbent whose particle diameter is 5–7 µm. Comparison of the characteristics of the plates for TLC and HPTLC is given in table.22. Monolithic sorbents are a new generation of stationary phases that can be used and in planar chromatography are obtained by direct copolymerization of methacrylic polymers, for example, a copolymer of glycine methacrylate and ethylene dimethacrylate. Monolithic stationary phases do not contain particles, and the role of the separation space is played by the surface and volume of the flow channels (pores). The macroporous structure of monolithic sorbents contains at least two types of pores: macropores and mesopores. The advantages of such carriers are a noticeable increase in the speed and efficiency of separation, since they do not have the usual diffusion limitations of interfacial mass transfer.

Table 1. Comparison of characteristics of classical (TLC) and high performance (HPTLC) thin layer chromatography plates.

Characteristics
Average particle size, microns
Layer thickness, microns
Number of samples
Solvent front run length, mm
Separation time, min
The amount of solvent, ml
Detection limit, ng
absorption

fluorescence

Main types of sorbents used in TLC

silica gel

polar adsorbent, contains active silanol and siloxane groups, it is used to separate compounds of different polarity.

Aluminium oxide

a polar adsorbent with a heterogeneous surface, contains active OH groups, has markedly pronounced proton-acceptor properties; it is used to separate aromatic hydrocarbons, alkaloids, chlorohydrocarbons, steroids

Florosil - the main magnesium silicate, occupies an intermediate position between aluminum oxide and silica gel; useful for separating flavanoids, steroids and acetylated hydrocarbons

Polyamides - a group of polar sorbents with mixed

separation mechanism: the carboxamide group is responsible for the adsorption mechanism, the methylene units are responsible for the distribution mechanism. These sorbents are used to separate food dyes, flavonoids, tannins, nitrophenols, alcohols, acids.

Modified silica gels with grafted groups (amino, cyano, diol-, C 2 -, C g -, C 1g -) of different polarity.

An important characteristic of the sorbent is its activity; it depends on the water content and decreases with increasing water content in the sorbent.

The choice of a sorbent is of great importance for the successful separation of mixtures of substances. First of all, it is necessary to proceed from the properties of the compounds to be separated: their solubility (hydrophilicity, hydrophobicity), content and nature of functional groups. Saturated hydrocarbons adsorb weakly or not at all on silica gels and alumina. INTRODUCTION of double bonds, especially conjugated ones, increases the adsorption capacity of the compounds.

Functional groups further enhance the ability of substances to adsorb. The adsorption capacity of functional groups increases in the following order:

CH=CH<ОСНз<СООR

Various methods are used to quantify the content of a substance in chromatographic zones:

1. Determination with the removal of the chromatographic zone from the plate can be carried out in two ways: by transferring the chromatographic zone together with the sorbent or by extracting the chromatographic zone from the sorbent layer.

2. Determination of compounds directly on the plate by visual comparison of the sizes of spots and their color with the corresponding parameters of spots of standard samples

3. The densitometry method, which improves the accuracy of the determination results, is based on scanning chromatograms in visible and UV light using "chromatographic spectrophotometers" densitometers. Densitometers make it possible to measure the absorption of light by a substance on a chromatogram in transmission or reflection mode, as well as fluorescence and its quenching. The transmission mode is available only if the substance under study has an absorption band in the visible region of the spectrum. In the UV region, registration in the transmission mode cannot be performed due to the intrinsic absorption of silica gel and the chromatogram substrate.

4. The video densitometer method is a relatively new method for the quantitative processing of chromatograms. The principle of the method is to enter a chromatogram image into a computer using a video camera or a digital camera, followed by a comparison of the spot intensities of standard and analyte compounds. The video densitometer includes a lighting unit, a video camera with a video capture card or a scanner, a personal computer with the installed Windows operating system and the corresponding software. In Russia, such complexes are produced by STC "Lenchrome" (St. Petersburg) - densitometer "DenScan-O4" and "Sorbpolimer" (Krasnodar) densitometer "Sorbfil". The program for processing chromatographic data allows you to perform the following functions: enter images of chromatograms and save them with high quality and resolution; to select on the input chromatogram image a working area, where the image will be further processed; produce

automatic or manual search for spots; carry out the processing of spots, convert them into the form of chromatographic peaks, calculate the values of R r and peak areas; measure the content of the substance in the analyzed spots (in relative units); enter concentration values to build calibration dependencies: linear interpolation; linear approximation more than, through two points; quadratic interpolation; automatically calculate the content of the substance in the analyzed spots according to the entered calibration values; present the results in the form of printed documents. 1-3

Quantitative processing of a spot in video densitometry is carried out according to two characteristics: by the area of the spot and its “volume” in space, with all this, brightness (spot color intensity) is used as the third coordinate (Fig. 1).

Rice. 1. View of the spatial distribution of brightness in the spot area:

Ai,j - value of the brightness level of the spot point; Bi,j is the value of the brightness level of a point on the base surface.

5. Densitometry with a flatbed scanner with software for processing chromatograms that practically does not differ from standard programs used for video densitometers, but at a significantly lower cost. In this case, scanning provides a clearer image of chromatographic zones, which can be explained by the reduced effect of uneven illumination of the analyzed objects than in the case of a video densitometer.

Application for solving practical problems. The use of those is especially effective for the preliminary separation (by classes, groups, types of substances) of the components of complex mixtures of organic pollutants in water, soil and air. Individual identification using only those is difficult due to the lack of highly sensitive and selective detectors, in addition, the determination of target components is less accurate than in the case of GC and HPLC. TLC is often used at the first stage of analysis to separate complex and multicomponent mixtures of organic compounds into separate simpler groups, and only then a more detailed study of these groups is carried out using “finer” methods (GC, HPLC, NMR, IR, or mass spectrometry).

The use of TLC in the analysis of contaminated fresh and sea water opens up wide possibilities for preparative separation before other methods, separation of desired impurities and additional identification. TLC is used to detect and

semi-quantitative determination of substances of different nature: surfactants, hydrocarbons, PAHs, phenols, pesticides.

To determine non-ionic surfactants in waste and river waters, plates with a layer of silica gel or Kiselgel o.d. are used. A chloroform extract of surfactants is applied to the plate and they are separated using mixtures of ethyl acetate: water: acetic acid as the mobile phase. Spots are detected by spraying with a mixture of: Burger's reagent: phosphoric acid: ethanol 5% solution of BaCI 2 .2H 2 0 (10:1:10:5). Surfactants show up as pink spots. The method allows to determine in water from 0.1 to 1.0 mg/l of nonionic surfactants. Under these conditions, ionic surfactants are extracted from wastewater, but they move along with the solvent front and do not appear.

Many methods for the determination of phenols have been proposed. Chlorophenols are separated on plates with aluminum oxide by repeated elution with benzene or on silica gel plates by elution with a mixture of benzene and petroleum ether (1: 1). Phenols are determined by the manifestation of a 2% solution of 4-aminoantipyrine (detection limit 0.5 μg / l) or by fluorescence at 254 nm (up to 0.5 μg of phenols). The second option for determining phenols is separation in the form of: antipyrine, 4-aminoantipyrine derivatives or with p-nitrophenyl azo dyes.4-6

CHAPTER 2. STATUS AND PROSPECTS OF THE USE OF MODERN INSTRUMENTAL METHODS OF PESTICIDE ANALYSIS IN UKRAINE

The increase in the scale and range of pesticide use in agricultural practice continues to stimulate the development and use of methods of analytical chemistry of low concentrations of toxic organic substances for the analysis of environmental objects, agricultural raw materials, feed and food. Determination of pesticide residues in these media is not of independent importance, but is a necessary part of the general information to achieve an adequate assessment of the risk associated with the use of pesticides. Risk assessment in the past has been mainly related to human safety and for this reason the determination of pesticide residues has focused mainly on agricultural raw materials and foodstuffs. In recent years, increasing attention to the impact of pesticides not only on humans, but also on their environment, requires much more information on the residual amounts of not only the pesticides used, but also the products of their destruction and metabolism in various environments. The study of pesticide residues now includes all types of agricultural raw materials, feed and food, water, air and soil. This, combined with the introduction of pesticide preparations with low consumption rates into agricultural technologies (<10 г/га) требует принципиально новых подходов и методов для идентификации и количественного определения остатков пестицидов в различных средах.

Taking into account the amount of information that must be obtained from the analysis of various matrices and media, a method for performing measurements (MPM) of pesticide residues should meet most or all of the following requirements:

Ensure reliable separation of the analyte from interfering impurities;

Provide unambiguous identification of the analyte;

Have a low limit of quantitation;

Have a short analysis time;

Have a low cost;

Ensure a reasonable degree of accuracy and correctness of the results;

Ensure the reliability of the results.

The desire of method developers to satisfy these requirements as fully as possible is one of the main incentives for improving the MIM. The modern MVI, based on instrumental methods of analysis, is divided into the following stages:

Extraction of analyzed pesticides and their metabolites;

Purification of the resulting extract;

Possible obtaining of derivatives of analyzed pesticides and products of their destruction and metabolism;

Chromatographic separation

Determination (detection) of analyzed substances.

The extraction method used in MVI should ensure the quantitative and selective extraction of analytes, i.e., the maximum extraction of analytes from the analyzed matrix against the background of the least possible extraction of co-extractive (interfering) substances. Otherwise, a more complicated stage of purification of the resulting extract will be required, which will inevitably lead to losses of analytes and an increase in the total analysis error. As a result, there is a general trend in pesticide residue analysis today to use extraction methods that are easy to automate, reduce the number of manual steps and the amount of organic solvents used, and enable the analysis of a large number of samples. These requirements are met by solid phase extraction (SPE), which is an alternative to traditional liquid-liquid extraction and which allows you to combine sampling with concentration. The use of ready-made commercially available cartridges (cartridges) for SPE greatly simplifies the procedure for preparing samples for analysis compared to traditional methods. SPE is used not only in water analysis, but also in the analysis of soil, fruits, vegetables and other food products. From the extracts of these matrices obtained using low-polarity and non-polar organic solvents, pesticides are then concentrated on molecular sorbents due to dipole-dipole interactions or the formation of hydrogen bonds. For these purposes, cartridges filled with silica gel, florizil or aluminum oxide are used. We have systematically studied the process of dynamic sorption of trace amounts of pesticides of various classes on a macronet "supercrosslinked" copolymer of styrene with divinylbenzene (polysorb). It is interesting to note that in the joint project SMT4-CT96-2142 of seven European research centers of France, Belgium, Germany, the Netherlands, Spain and Portugal, which started in 1997 and the subject of which was the development of a method for determining of multiple pesticide residues in drinking water using SFE, which allows to control pesticides in water at a level of 0.1 µg/l (in accordance with the requirements of the European Drinking Water Directive 80/778/EEC), nine sorbents from different companies based on C18- converted oh phase and SDB-1 . As a result of these studies, it was found that the most suitable sorbent for SPE of pesticides from water was SDB-1, a sorbent based on a copolymer of styrene with divinylbenzene, the effectiveness of which for these purposes was established by us in the early 80s of the last century.

In recent years, for the extraction of pesticides from various matrices, sphere-critical fluid extraction (SFE) has been used, which is considered as an alternative to conventional liquid extraction in a Soxhlet apparatus. Carbon dioxide, nitric oxide, and mixtures of carbon dioxide and nitric oxide with methanol and toluene are used as supercritical fluids. Under supercritical conditions (temperature 40 °C, pressure 300 atm), the solvating properties of carbon dioxide are similar to those of freons or hexane. One of the main advantages of SFE is that, with all this, residues of various pesticides and products of their destruction and metabolism are extracted from the analyzed matrices, which are not extracted by traditional methods, even when extraction is carried out in a Soxhlet apparatus. The instrumentation of SPE makes it possible to fully automate this process. Ukrainian analytical chemists working in the field of pesticide analysis have yet to get acquainted with this powerful tool for extracting pesticide residues from soil, plant material and animal tissues, which allows extraction of a large number of samples. The efficiency of SPE for the analysis of such supertoxicants as polychlorinated dibenzodioxins and polychlorinated dibenzofurans is particularly impressive.

As a method for purifying extracts in the analysis of pesticide residues, gel chromatography is often used today, either as a standalone method or as a step in a multi-step purification operation. This purification method is especially effective in the analysis of matrices containing a large amount of lipids. Gels operating in organic solvents have received the greatest use for this purification method. Automated installations have been developed that allow the purification of a large number of samples without any attention from laboratory personnel. The effectiveness of this purification method was first demonstrated by us in domestic studies for the purification of extracts from rice containing Saturn and Prefix herbicides using gels formed by weakly crosslinked copolymers of styrene with divinylbenzene, which swell well in low-polar and non-polar organic solvents.

Gel chromatography is an indispensable step in the multi-stage purification operation in the development and use of so-called methods for the determination of multiple residues (multiresidue) of pesticides. The increase in the number of pesticides used and the sources of their entry into environmental objects, agricultural raw materials and food causes a significant increase in the volume of chemical-analytical studies. Naturally, it is economically unprofitable and inconvenient to use a separate MIM to determine each pesticide in each analyzed matrix. Much more attractive are such methodological approaches that make it possible to cover the entire amount of pesticides used in agricultural practice by several MVIs. This approach has a number of important advantages: firstly, the total analysis time is significantly reduced; secondly, the total number of pesticides and their metabolites that can be determined by these methods increases dramatically and, thirdly, these methods can be quickly adapted, if necessary, to new analyzed matrices and to new pesticides. At present, abroad, to control the content of pesticides, only methods for determining multiple pesticide residues are used, which allow the determination of almost all pesticides that are used in agricultural practice in one sample of agricultural raw materials, food, water, soil or air. For example, the method for the determination of multiple residues AOAC 990.06 allows the determination of 29 organochlorine pesticides in one sample of drinking water. The AOAC 991.07 Multiple Residues Method is designed to determine 44 nitrogen and organophosphate pesticides in a single drinking water sample. The German Ministry of Health's Multiple Residue Method S 8 is designed for the determination of 91 chlorine, phosphorus and triazine pesticides in a single fruit or vegetable sample. The technique for the determination of multiple residues S 19 (Germany) allows the determination of 220 chlorine-, phosphorus- and nitrogen-containing pesticides in one soil sample. The methodology of the European project SMT4-CT96-2142 makes it possible to determine in one sample of drinking water 38 pesticides that are a priority for the countries that developed the methodology.

Unfortunately, until now in Ukraine, when developing MVIs intended to control the content of pesticide residues, an approach is used that was formed in the bowels of the State Commission for Chemical Pest Control, Plant Diseases and Weeds of the former USSR, and which consists in the need to develop a separate methods for each pesticide and each matrix analyzed. This development is based on the methods presented by the pesticide developer company along with a report on the validation of the presented methods by an independent laboratory and the results of field tests to determine pesticide residues in crops, soil, water and air of the working area. These methodologies are presented by the pesticide product development company only for passing the state registration of the pesticide in Ukraine in order to show that the data on pesticide residues in crops, soil, water and air of the working area, which the company represents, were obtained using validated methods. Thus, the MVI provided by the pesticide product development company serve only for the purposes of the state registration of the pesticide and are not MVI, which are used in the pesticide product development country to control the content of pesticide residues in agricultural raw materials, food products and environmental objects. The prerogative of the development of MVI, designed to control the content of pesticide residues in various media, is assigned abroad not to pesticide manufacturers, but to ministries and departments that are responsible for a particular area of control. For example, in the US, these are the Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA).

Thus, in order to develop a modern strategy for the use of MVI to determine pesticide residues in Ukraine, it is necessary to clearly distinguish between MVI, which are necessary for the purposes of state registration of pesticides, and MVI, which are intended for state sanitary and epidemiological supervision of the use of pesticides. For the purposes of state registration of pesticides, the following approach to the development of MIM is economically and methodologically justified: one pesticide - one crop/environment - one MVI. The development of such MVIs is based on the methods presented by the pesticide product development companies. The MVI developed in this way are used in the determination of pesticide residues in agricultural raw materials, soil, water and air of the working area only during pre-registration state tests of pesticides. For the purposes of state sanitary and epidemiological supervision over the use of pesticides, of course, MVI is necessary, the development of which is based on the principle of determining multiple pesticide residues in one sample. The use of such MVI will significantly reduce the cost of both their development and subsequent sanitary and epidemiological surveillance of the use of pesticides. Currently, the issue of resuming the operation of the pesticide monitoring system is being considered, which at one time (1984-1991) was developed at VNIIGINTOKS (now the Institute of Ecohygiene and Toxicology named after L.I. . Such monitoring should be based only on methods for the determination of multiple pesticide residues. We have analyzed the chemical-analytical aspects of the functioning in the past of a unified system for monitoring pesticide residues in agricultural raw materials, food products and environmental objects, outlined ways to modernize this system and methodological approaches for developing methods for determining multiple pesticide residues in fruits, vegetables and water.

A number of pesticides used in Ukrainian agriculture cannot be subjected to direct gas chromatographic determination due to their low volatility or insufficient thermal stability. In order to make it possible to determine these compounds using GC, they are converted into various derivatives. Such an operation usually increases the volatility and reduces the adsorption of the chromatographed compounds on solid carriers, increases their thermal stability and improves separation. In some cases, with all this, a significant increase in the sensitivity of detection of the obtained derivatives is also achieved. All this is the subject of reactive gas chromatography. For the first time in domestic research, we have shown the effectiveness of using reaction gas chromatography in the analysis of pesticides by the example of determining the residual amounts of herbicides - derivatives of phenoxyalkanecarboxylic acids (2,4-D, 2,4-DM) in food products. Since then, the method of reaction gas chromatography has been widely used in the laboratories of the Institute when conducting state tests of pesticides and carrying out state sanitary and hygienic examination.

The HPLC method has demonstrated certain advantages in the joint determination of pesticides and their metabolites in one sample. This is especially true for those pesticides that cannot be determined by GC due to their thermal instability, high polarity and low volatility. The use of HPLC in the analysis of pesticides eliminates the laborious operation of derivatization. The Institute was one of the first in Ukraine to start using this method for the determination of pesticides. At present, HPLC is a routine method of analysis in many laboratories of the Institute. This method is especially widely used during the state sanitary and hygienic examination of food products.

Listing the chromatographic methods that are used in the analysis of pesticide residues, one cannot fail to mention the thin layer chromatography (TLC) method, which was discovered in 1938 by Ukrainian scientists N.A. Izmailov and M.S. Schreiber. Semi-quantitative TLC is still an inexpensive and effective method for separating, identifying and semi-quantifying pesticide residues. It was the semi-quantitative version of TLC that played a big role in the formation of the chemical analytical service of the Ministry of Health of Ukraine for monitoring the content of pesticide residues in food and environmental objects, when GC and HPLC methods were not yet available for wide use. This was largely due to the work carried out within the walls of the Institute. Currently, TLC in the analysis of pesticide residues is mainly used as an alternative analytical method to confirm the correct identification of pesticides obtained using GC and HPLC methods. TLC is also an indispensable tool in the analysis of pesticide residues, when it is necessary to check a very large number of food or environmental samples for the presence of pesticides. In such cases, a screening methodology is usually applied. All samples that give a "positive" reaction are further analyzed by some more specific instrumental method (GC, HPLC, GC/MS, LC/MS), while all negative screen results are accepted as final without any verification. The Institute has a set of equipment for quantitative TLC (KAMAG, Germany). Nevertheless, the prospects for further use of TLC in the analysis of pesticides should primarily be associated with a semi-quantitative version of this method. There is no alternative to this.

Each stage of the use of pesticides in world agricultural practice from the late 40s of the last century to the present can be characterized by its own chemical and analytical problems. However, one problem in the analysis of pesticide residues remains unchanged - the need to constantly reduce the limits of quantitative determination (limit of quantitafication, LOQ) of pesticides. Achieving very low limits of quantitative determination when using MVI is accompanied by a decrease in the level of reliability (identification reliability) of the analysis result. Often, in order to achieve very low limits of quantitation, it is necessary to use a complex multi-step purification procedure and a derivatization step so that highly selective and highly sensitive detectors (ECD, TID) can be used. In this case, this is inevitably accompanied by losses of the analyte during these operations, which leads to an increase in the analysis error. In addition, the variability of the composition of the analyzed matrix from sample to sample also contributes. In this regard, an analytical chemist cannot always satisfy the desire of a hygienist and toxicologist to have MVI with very low limits of quantitative determination due to the technical capabilities of the instruments used and the methodological limitations of the MVI being developed. When developing MVI, the analytical chemist should focus his efforts not only on achieving low limits of quantitative determination of the analyzed pesticides, but not lose sight of the more important aspects of the analysis of pesticide residues: reliability of identification and reproducibility of results. It is known that today in Ukraine in some agricultural crops and food products the content of pesticides is not allowed (the so-called zero tolerances) or is at the level of the limit of detection (LOD), i.e. any detectable pesticide residues are considered unacceptable. In such cases, the reliability of identification of the pesticide is of paramount importance, and not the exact quantitative determination of its content, since the very fact of the detection of a pesticide is the basis for a ban on the use of agricultural raw materials or food products. In these cases, the use of a semi-quantitative TLC variant is fully justified, provided that, with all this, a reliable identification of the pesticide being determined is achieved.

Understanding the importance of issues related to improving the reliability of identification of analytes in the analysis of pesticide residues, we undertook systematic studies on the study of intermolecular interactions of chlorine- and nitrogen-containing pesticides under gas and liquid chromatography conditions. At the same time, the existence of correlation dependences between the retention parameters of members of the homologous series of sorbates obtained using chromatographic methods with different sorption mechanisms was established for the first time. The effectiveness of using such dependencies to improve the reliability of pesticide identification was demonstrated using the homologous series of chloralkanecarboxylic and chlorophenoxyalkanecarboxylic acids and their esters, chlorophenols, substituted phenylureas, nitrophenols and nitrophenolic compounds, substituted benzoic acids, s-triazines, and thiocarbamic acid esters as an example. 9

Chapter 3. GUIDELINES FOR THE DETERMINATION OF ORGANOCHLOROGENIC PESTICIDES IN WATER, FOOD, FEED AND TOBACCO PRODUCTS BY THIN LAYER CHROMATOGRAPHY

This technique has been tested and recommended as an official group of experts under the State Commission for Chemical Pest Control, Plant Diseases and Weeds under the Ministry of Agriculture of the USSR.
These Guidelines apply to the determination of the content of DDT, DDE, DDD, hexochlorane, aldrin, keltan, heptachlor, methoxychlor, dactal, tedione and ethersulfonate in water, soil, wine, vegetables, fruits, mushrooms, grain, compound feed, root crops and green fodder, fish, meat, meat products, internal organs, milk and dairy products, animal fat, butter and vegetable oils, cakes, meal, husks, honey, sugar, eggs and egg products, as well as in tobacco products.

The principle of the method. The method is based on the chromatography of chlorine-containing pesticides in a thin layer of aluminum oxide, silica gel or Silufol plates in various systems of mobile solvents after their extraction from the studied samples and purification of the extracts. The mobile solvent is n-hexane or n-hexane mixed with acetone. Places of localization of drugs are found after spraying the plates with a solution of silver ammonia, followed by ultraviolet irradiation or after irradiating Silufol plates containing o-tolidine with ultraviolet light.

Reagents and solutions

Acetone, chemically pure, GOST 2603-71

Ammonia water chemical pure, GOST 3760-64

Aluminum oxide 2 tbsp. activity for chromatography, h, MRTU 6-09-5296-68. Sift through a 100 mesh sieve.

Aluminum oxide impregnated with sulfuric acid. Two weight parts of aluminum oxide (or silicon oxide) are placed in a porcelain mortar, poured with one volume part of sulfuric acid and mixed thoroughly. The mixture is prepared immediately before the preparation of columns for the purification of extracts from samples of meal, cake, husk

Chemically pure benzene, GOST 5955-68

N-hexane pure, MRTU 6-09-2937-66

Potassium oxalate, analytical grade, GOST 5868-68

Calcium sulfate chda, GOST 3210-66. Dry for 6 hours in an oven at 160 degrees. C. Screen through a 100 mesh sieve.

Silicon oxide for phosphors h, MRTU 6-09-4875-67

Sodium sulfate anhydrous h, GOST 4166-66

Sodium carbonate, chemically pure, GOST 4201-66, 0.5 n. solution

Sodium chloride, chemically pure, GOST 4233-66, saturated solution

Petroleum ether (bp 40 - 70 degrees)

Hydrogen peroxide, chemically pure (30% aqueous solution), GOST 10929-64

Developing reagents:

Developing reagent N 1. 0.5 g of silver nitrate is dissolved in 5 ml of distilled water, 7 ml of ammonia are added and the volume of the solution is adjusted to 100 ml with acetone; 0.2 ml of hydrogen peroxide can be added to the finished solution. The solution should be stored in a stoppered flask in a dark place for 3 days. On a 9 x 12 cm plate, 8 - 10 ml of solution is consumed. Developing reagent N 2. 0.5 g of silver nitrate is dissolved in 5 ml of distilled water, 10 ml of 2-phenoxyethanol is added and the volume of the solution is adjusted to 200 ml with acetone, then 6 drops of 30% hydrogen peroxide are added.

Silver nitrate pure, GOST 1277-63

Sulfuric acid pure, GOST 4204-66

Silica gel ASK (Voskresensky chemical plant, Moscow region)

Silica gel KSK, sifted through a 100 mesh sieve.

Standard Samples:

DDT, DDD, DDE, aldrin, HCCH isomers, heptachlor, methoxychlor, keltan, ethersulfonate, dactal, tedion hch.

Standard solutions: Dissolve 10 mg of the appropriate pesticide in a 100 ml volumetric flask in n-hexane and make up to the mark with this solvent. Standard solutions should be stored in glass bottles with ground stoppers in the refrigerator.

Glass wool, purified conc. sulfuric acid, washed with distilled water and dried o-Tolidine h, MRTU 6-09-6337-69, 1% solution in acetone2-phenoxyethanol

Ethyl alcohol, rectified, TU 19-11-39-69

Chloroform, chemically pure, GOST 200-15-74

Carbon tetrachloride, chemically pure, GOST 20228-74

Ethyl ether (for anesthesia), USSR Pharmacopoeia

Sodium sulfate, 2% aqueous solution

Sodium sulfate, saturated solution

2.4. Cutlery and utensils

Water bath, TU 64-1-2850-76

Vacuum-rotary evaporator, IR TU 25-11-310-69 or solvent stripper, MRTU 25-11-67-67

Funnels chemical, to dia. 6 cm, GOST 86-13-64

Dividing funnels, capacity 100, 250, 500 ml, GOST 10054-75

Buechner funnels, GOST 9147-69

Homogenizer or tissue grinder

Spray chamber, TU 25-11-430-70

Chamber for chromatography, size 150 x 200, 105 x 165 mm, GOST 10565-63

Bunsen flasks, TU 25-11-135-69

Volumetric flasks, capacity 50, 100 ml, GOST 1770-74

Flasks nsh, capacity 100, 250, 500 ml, GOST 10394-63

Round bottom flasks nsh, capacity 150, 250, 500 ml, GOST 10394-63

Micropipettes, GOST 1770-74 (for applying standard solutions)

Pipettes or syringes for sample application

Pipettes with a capacity of 1, 5, 10 ml, GOST 1770-74

Shaking device, MRTU 2451-64

Glass plates 9 x 12, 13 x 18 cm

Glass atomizers for spraying plates

100 mesh sieve (hole diameter 0.147 mm)

Glass chromatographic columns (diameter - height), 20 x 400, 15 x 150

Mercury-quartz lamp

Measuring cylinders with a capacity of 25, 50, 100, 250, 500 ml, GOST 1770-74

Evaporating cups N 3, N 1, GOST 9147-69

Preparation of plates for chromatography

Thoroughly washed with a chromium mixture, a soda solution, distilled water and dried, the plate is wiped with ethyl alcohol or ether and

covered with sorption material. The mass is prepared as follows:

a) 50 g sifted through a 100 mesh sieve. aluminum oxide is mixed in a porcelain mortar with 5 g of calcium sulfate, 75 ml are added

distilled water and mix in a mortar or flask until a homogeneous mass is formed. 10 g of the sorption mass is applied to a 9 x 12 cm plate (20 g is applied to a 13 x 18 cm plate) and, shaking, is evenly distributed over the entire plate. The plates are dried at room temperature for 18 - 20 hours, you can dry them for 20 minutes at room temperature, and then 45 minutes in an oven at a temperature of 110 degrees. C.

b) 35 g of KSK silica gel, sifted through a 100 mesh sieve, mixed with 2 g of calcium sulfate and 90 ml of distilled water and stirred in a mortar or flask until a homogeneous mass. Apply to the plates and dry as above. The serving is for 10 plates.

If thin silica gel sheets darken after being irradiated with UV light, the silica gel should be cleaned of impurities before use. To do this, silica gel is poured for 18 - 20 hours with dilute hydrochloric acid (1: 1), the acid is drained, the silica gel is washed with water and boiled in a round-bottom flask for 2 - 3 hours with dilute nitric acid (1: 1), washed with running tap water, then with distilled water to a neutral reaction of washing water, dried in an oven for 4 - 6 hours at a temperature of 130 degrees. The silica gel is crushed and sieved through a 100 mesh sieve.

Plates for chromatography "Silufol" UV-254 produced by Czechoslovakia are impregnated with o-tolidine before use. To do this, each plate is lowered by 0.5 cm into a 0.1% solution of o-tolidine in acetone, poured into the chromatography chamber. After the solvent front rises to the upper edge of the plate, it is removed and dried in air, avoiding direct sunlight. After that, the plates are ready for use. Plates impregnated with o-tolidine are stored in a desiccator. Used in feed analysis.

Plates "Silufol" UV-254 produced by Czechoslovakia washed with distilled water in a chromatographic chamber, dried in air and immediately before use, activated in an oven at a temperature of 65 degrees. within 4 minutes. Preparation of chromatographic columns for purification of extracts

Chromatographic column for purification from milk fat. At the bottom of a chromatographic column (20 x 400 mm in size) place glass wool or 500 mg of fat-free cotton wool. Then, ASA silica gel is poured into the column (75 ml for purification of extracts from pork fat samples and 70 ml for all other samples) and the silica gel is compacted by tapping on the column. The column is washed with 50 ml of n-hexane or petroleum ether, and the solvent that has passed through it is discarded. After that, the column is ready for chromatographic purification of extracts from samples of fish, meat and meat products, milk and dairy products, honey, eggs, etc.

Chromatographic column for purification of extracts from meal samples (not enriched with lipids) and husks.

The chromatographic column is filled to a height of 1 cm with glass wool, then sieved aluminum oxide (I) is added to the column with a layer of 2.5 cm or silicon oxide - 3.5 cm. Next, lumps of aluminum oxide (silicon) impregnated with sulfuric acid , height of layer (II) 2.5 cm. Each layer is successively washed with hexane (total 20 - 30 ml).

For the analysis of cakes and meals enriched with lipids, the aluminum oxide layer should be increased to 5 cm (I) and 3 cm (II), respectively, in the case of using silicon oxide, 6 cm (I) and 3 cm (II).

Water, wine. A 200 ml sample is placed in a separating funnel and the pesticides are extracted by shaking for 3 minutes with n-hexane or petroleum ether in three portions of 30 ml or diethyl ether in three portions of 50 ml. 10 g of anhydrous sodium sulfate are poured into the combined extracts or filtered through a funnel filled 2/3 with sodium sulfate. The extracts are transferred to a solvent stripper and the solvent is distilled off to a volume of 0.2 - 0.3 ml. If necessary, the extract is cleaned with sulfuric acid.

Vegetables fruits. 20 g of the crushed sample is placed in a flask with a ground stopper and the pesticides are extracted three times for 15 minutes on a shaker with n-hexane or petroleum ether in 30 ml portions. The combined extracts are dried with anhydrous sodium sulfate, transferred to a solvent stripper, the solvent is distilled off to a volume of 0.2 - 0.3 ml and applied to the plate.

Grain, mushrooms. From the crushed samples, 20 g of grain, 50 g of raw or 10 g of dry mushrooms are taken and placed in flasks with ground stoppers. Extraction of pesticides is carried out three times on a shaker with n-hexane or petroleum ether in 30 ml portions. The combined extracts are transferred to a separating funnel, 10 ml of a saturated solution of anhydrous sodium sulfate in sulfuric acid is added and gently shaken several times. Separate the organic layer and repeat the treatment until the acid is colorless. The extract was washed with distilled water, dried with anhydrous sodium sulfate and the solvent is distilled off.

Apples, cabbage, grass, hay. 20 g of crushed apples, 20 g of cabbage, 40 g of grass and 20 g of hay are poured into 100 ml of acetone in a flask with a ground stopper. Shake for 2-3 minutes, add 20 ml of distilled water and cool on ice for 30 minutes. The extract is drained and filtered cold, the extraction is repeated. Acetone is distilled off from the combined water-acetone extracts, and the preparations are extracted from the aqueous residue with n-hexane in three portions of 10 ml for 10 minutes. Hexane extracts are purified with sulfuric acid saturated with anhydrous sodium sulfate. Dry with anhydrous sodium sulfate. The solvent is distilled off to a small volume and applied to the plate. If the purification is incomplete (after evaporation of the solvent, a white coating remains on the flask), the extract is evaporated dry, the residue is washed off with cold acetone 3 times in portions of 0.2 ml and immediately applied to the plate.

Compound feed. For research, take a sample of 40 g, moisten it in a flask with 60 ml of distilled water. The moistened sample is left overnight in a stoppered flask. Extraction of pesticides is carried out twice with 50 - 100 ml of a mixture of hexane - acetone 1:1 with shaking for 2 hours. The extracts are combined in a 500 ml separatory funnel, 50 ml of distilled water are added twice and, after separation of the layers, the lower aqueous layer is poured into another separatory funnel and the pesticides are extracted with 40 ml of hexane. The water layer is drained. Hexane extracts are combined, filtered through a funnel with a paper filter filled with 2/3 anhydrous sodium sulfate. The extracts are evaporated on a rotary evaporator to a volume of 20-30 ml or to dryness, then the dry residue is dissolved in 20-30 ml of hexane or petroleum ether. The extract is transferred to a separating funnel and purified with sulfuric acid as described above.

Meal, husk, cake. Samples: 15 g lipid-enriched meal or cake; 20 g of meal or husk not enriched with lipids is divided into equal parts and placed in flasks with a capacity of 100-250 ml with ground stoppers, poured with hexane (three volumes of hexane per one weight part of the meal), shaken on a shaking device for 30 minutes. The extract is filtered through a Buchner funnel without transferring the precipitate to the funnel. The indicated amount of hexane is refilled into the flask, shaken for 30 minutes, filtered, the precipitate is quantitatively transferred to a Buchner funnel with 30 ml of hexane (3 times 10 ml). The resulting extract is evaporated to 30 ml on a rotary evaporator or in a stream of air at a temperature not exceeding 40 degrees, the residue is divided into two equal parts and placed in the freezer of the refrigerator for 1 hour (at least). Each portion is passed through a separate alumina column at a rate of 2 ml/minute, wash the flask and column with 50 ml of chilled ethyl ether/hexane (15:85). This operation must be carried out without interruption, without leaving the next day. The purified extracts are combined and evaporated to a volume of 1 ml. The residue from the flask is transferred quantitatively with a micropipette using a rubber bulb into a 1 ml test tube, the flask and micropipette are washed 2-3 times with a small amount of hexane (0.3-0.5 ml in total), pouring it into the same test tube. The hexane is then carefully evaporated from the tube in a water bath at 50° to almost dryness (final volume approximately 2-3 drops). If the total volume of the extract and washing liquid exceeds 1 ml, then the extract is first evaporated, gradually adding washing liquid to it. If there is a white, ointment-like precipitate in the evaporated extract, add 5-6 drops of hexane to the test tube and place it for 15-20 minutes in the freezer of the refrigerator, then decanate twice with the same amount of hexane and again evaporate to a final volume of 2-3 drops.

In parallel with the studied samples, two model extracts are prepared. Each extract is obtained from one gram of pesticide-free meal (the ratio of dry matter and pesticide is the same as in the studied samples). In one of the extracts, before purification on the columns, the determined pesticides are added with a microsyringe (micropipette) in the amount of 3 μg, in the other - 0.75 μg. The evaporated test and model extracts are quantitatively applied to the plate using a micropipette or microsyringe, washing the tube three times with a small amount of hexane.

Fish, meat and meat products. Meat, meat products are passed through a meat grinder. The fish is cleaned of scales, internal organs and also passed through a meat grinder. 20 g of the sample is mixed with anhydrous sodium sulfate and placed in a flask with a ground stopper. Pesticides are extracted twice with a mixture of hexane - acetone or petroleum ether - acetone in a ratio of 1:1 in portions of 50 ml for 1.5 hours with shaking.

The extract is filtered through a funnel with a paper filter filled to 2/3 with anhydrous sodium sulfate, then the solvent is distilled off, the dry residue is dissolved in 20 ml of n-hexane and added to a column of ASA silica gel. After the extract has been absorbed into the sorbent, the pesticide is eluted with 110 ml of a mixture of benzene and hexane in a ratio of 3:8 in portions of 25–30 ml. The eluate is collected in a round bottom flask with a thin section with a capacity of 250 - 300 ml. 10 minutes after the last portion of the solvent has been absorbed, the sorbent is squeezed out with a pear. The eluate is distilled off to a volume of 0.1 ml and applied to a chromatographic plate.

In the event that meat or fish samples contain a large amount of fat, after evaporation of the first extractant (a mixture of acetone and hexane) and dissolution of the dry residue in hexane, the hexane extract should be purified with sulfuric acid, and then column purification, as described above.

Animal fat, eggs, egg powder. The fat is crushed in a meat grinder, the egg powder is thoroughly mixed, the eggs are separated from the protein, the yolk and protein are weighed, and only the yolk is taken for analysis. The final result on the content of organochlorine pesticides in the egg is given for the whole egg. The yolk is thoroughly mixed. 25 g of the prepared sample is poured into 50 ml of acetone, mixed and heated in a hot water bath until the solvent boils. The flask is cooled, 10 ml of chilled 2% sodium sulfate solution is added to it, stirred and cooled for 45 minutes in an ice bath. Then the acetone layer is poured into a round bottom flask through a layer of fat-free cotton wool. Extraction with acetone followed by freezing the fat is repeated two more times. Acetone is distilled off from the combined extracts on a rotary evaporator or in a solvent stripper (bath temperature not more than 70 degrees +/- 2 degrees) and extracted three times with petroleum ether in portions of 20, 10 and 10 ml. The duration of the first extraction is 1 hour, subsequent - 15 minutes. Petroleum ether is transferred to a separating funnel with 40 ml of 2% aqueous sodium sulfate solution, mix the contents for 2 minutes, allow the layers to separate and discard the aqueous phase. A few ml of saturated sodium sulfate solution can be added to improve layer separation. The operation of washing the extract is repeated two more times, after which the petroleum ether is poured into a beaker with 20 g of anhydrous sodium sulfate, the separating funnel is rinsed twice with 5 ml of petroleum ether. The dried extract is quantitatively transferred into a 50 ml measuring cylinder and the volume of the solution is adjusted to 30 ml with petroleum ether. Next, 30 ml of the extract is applied to an ASA silica gel column as described above. For pork fat samples, 75 ml of ASA silica gel are poured, for all other samples - 70 ml. Purification of extracts is carried out as described for meat samples. The eluate is collected in a 150 ml round bottom flask, the solvent is evaporated to a volume of a few drops and applied to a chromatographic plate.

Honey. 30 g of honey is mixed with 3 g of anhydrous sodium sulfate and the pesticides are extracted three times with hexane in portions of 30 ml each time for 15 minutes, carefully rubbing the honey with a glass rod in a narrow beaker. The extracts are combined and distilled off with hexane to a volume of 30 ml or less, then bringing the extract to 30 ml with hexane. 30 ml of the extract is added to a chromatographic column with ASA silica gel and the extract is purified and the solvent is evaporated as described above.

Sugar. From a sample of 50 g of sugar previously dissolved in water, the pesticides are extracted in a separating funnel with 250 ml of n-hexane. Extraction of pesticides is carried out three times with 50, 25 and 25 ml of solvent each time shaking for 5 minutes. The combined hexane extracts are purified from co-extractive substances (dyes, amino acids, lipids) by the sulfuric acid method.

Milk and dairy products. Samples can be prepared using one of the following methods.

First way. Cream, sour cream, milk and other whole milk products. For analysis, take 20 g of cream and sour cream, previously diluted with an equal volume of distilled water, 50 ml of milk, kefir, etc., add concentrated sulfuric acid (30 - 40 ml) until the sample is completely blackened. Cooled to 10 - 15 degrees. the solution is transferred to a separating funnel and the preparations are extracted with hexane 2 times in 25 ml portions. For complete extraction, the funnel is shaken for 2 minutes, then left for 30 minutes until the layers are completely separated. If an emulsion is formed, add 1-2 ml of ethyl alcohol. To the combined extracts in a separating funnel, add 10 ml of concentrated sulfuric acid saturated with sodium sulfate and gently shake several times. Purification is continued until colorless sulfuric acid is obtained.

Curd, cheese. 50 g of cottage cheese or 10 g of grated cheese are poured into 40 ml of hexane or petroleum ether, shaken continuously for 2-3 minutes and left for 30 minutes. The extraction is repeated. The combined separatory funnel extracts are purified with sulfuric acid as above.

The second way. Milk, kefir, curdled milk, koumiss and other whole milk products. 25 ml of the product is placed in a 300 ml separating funnel, 5 ml of potassium oxalate and saturated sodium chloride solution are added, stirred, 100 ml of acetone are added, shaken for 2 minutes. Add 100 ml of chloroform and shake for 2 minutes. The funnel is left until the layers are completely separated. The upper phase is discarded, and the lower phase is poured into a round-bottom flask with a thin section and the solvent is evaporated to dryness. The residue is washed off with 30 ml of hexane.

Condensed milk, 10 - 20% cream. To 10 g of the product, add 10 ml of a saturated sodium chloride solution and pour into a separating funnel with a capacity of 150 ml. 40 ml of acetone are added to the mixture, shaken for 2 minutes, 60 ml of chloroform are added, shaken for 2-3 minutes and left until the phases separate. Then proceed as in the determination of pesticides in milk.

Condensed dairy products. 10 g of the product is placed in a glass, pour 10 ml of water at a temperature of 45 - 50 degrees. C, mix and transfer to a 150 ml separating funnel, add 5 ml of potassium oxalate. The contents of the funnel are mixed, 80 ml of acetone is added and shaken for 2-3 minutes. Add 100 ml of chloroform and shake for 5-7 minutes. After separation of the phases, the lower phase is poured into a round bottom flask, the solvents are distilled off, and the dry residue is dissolved in 30 ml of petroleum ether. Dry dairy products. 3 g of dry dairy products (cream 2 g) are poured into a glass, 15 ml of distilled water is poured with a temperature of 40 - 45 degrees. C, stir and transfer to a separating funnel with a capacity of 300 ml, pour 5 ml of potassium oxalate and saturated sodium chloride solution. The contents of the funnel are stirred, 80 ml of acetone are added and shaken for 3-5 minutes, 100 ml of chloroform are added, shaken for 5 minutes and left for 3-5 minutes (until the phases separate). The lower phase is poured into a round bottom flask, the solvent is distilled off, and the residue is washed off with 30 ml of hexane. Sour cream, 30 - 40% cream. Weigh 5 g of the product into a beaker, add 10 ml of saturated sodium chloride solution and transfer to a separating funnel with a capacity of 150 ml. The glass is washed with 40 ml of acetone, the washings are transferred to a separating funnel, which is shaken for 2–3 minutes, 70 ml of chloroform is added and shaken for 2 minutes. The funnel is left for a few minutes until the phases separate, the lower phase is poured into a flask to distill off the solvents, the solvent is distilled off, and the residue is washed off with 30 ml of hexane.

Curd, cheese. 10 g of cottage cheese or grated cheese is triturated with 10 ml of a saturated sodium chloride solution and transferred to a separating funnel for 250 - 300 ml. Add 80 ml of acetone, shake for 2 minutes, add 100 ml of chloroform and shake again. The lower phase is used for analysis after distillation of the solvents, dissolving the residue in 30
ml of hexane. Further, extracts from milk and dairy products samples are purified from milk fat, prepared according to the second method. To do this, 30 ml of the extract is applied to a column with 70 ml of ASA silica gel. After the extract has been absorbed into the sorbent, the pesticide is eluted with 110 ml of a mixture of benzene and hexane in a ratio of 3:8 in portions of 25–30 ml. The eluate is collected in a 250-300 ml round bottom flask. 10 minutes after the last portion of the solvent has been absorbed, the sorbent is squeezed out with a rubber bulb. After purification, the solvents are distilled off under vacuum.
Butter. Melt 20 g of butter in a water bath in a round-bottom flask, add 50 ml of acetone, mix thoroughly until the fat dissolves, add 10 ml of ice-cold distilled water and cool on ice until the fat solidifies (about 30 minutes). Drain the acetone extract, and the procedure is repeated 2 more times. From the combined extracts in a round bottom flask, acetone is distilled off on a water bath. Pesticides are extracted from the remaining aqueous extract with hexane in three portions of 10 ml for 5 minutes. The combined hexane extracts in a separating funnel are treated with sulfuric acid with sodium sulfate. The purified extract is dried with anhydrous sodium sulfate and evaporated. The soil. To samples of air-dry soil (10 g) placed in 250 ml conical flasks, add 10 ml of a 1% aqueous solution of ammonium chloride and leave closed for a day. Then a mixture of 30 ml of acetone and 30 ml of hexane is added and the flasks are shaken for an hour on a shaking device. The contents of the flasks are transferred to centrifuge tubes. After centrifugation, the liquid part is poured into conical flasks, the soil is transferred to the original conical flasks with 10 ml of 1% ammonium chloride solution and 30 ml of acetone, 30 ml of hexane are added and extraction is carried out for another 30 minutes. The extracts are then combined. 180 ml of distilled water is added to the combined extracts in a separating funnel, gently shaken for 5-7 minutes, the liquids are allowed to separate and the lower aqueous layer is poured into a conical flask. The hexane layer is passed through anhydrous sodium sulfate (a tablespoon or 30 - 40 g of sodium sulfate). Pesticides are extracted from the water-acetone layer twice more with 15 and 10 ml of hexane, which is then dried over the same sodium sulfate. The hexane extracts are combined. The concentration of the extracts is carried out either on a rotary vacuum evaporator, or at a bath temperature of not more than 40 degrees. C and distillation time 9 - 11 minutes, or from flasks with an L-shaped outlet at a water bath temperature of 72 - 75 degrees. C.

Purification of concentrated hexane extracts from soil samples is carried out with sulfuric acid, as described above for other samples, and the solvent is evaporated. Tobacco and tobacco products. 5 g of tobacco is placed in a 500 ml glass beaker, poured with 50 ml of concentrated sulfuric acid and thoroughly stirred with a glass rod until the sample is completely uniformly charred. After 10 - 15 minutes, 25 ml of hexane are added to the flask, the contents are thoroughly stirred and 20 ml of carbon tetrachloride are added. Extraction of pesticides from the sample is carried out three times for 15 minutes, after which the extract is sequentially transferred to a separating funnel for single or double additional purification with sulfuric acid.

Chromatography.

On a chromatographic plate at a distance of 1.5 cm from its edge, the test sample is applied at one point with a syringe or pipette so that the spot diameter does not exceed 1 cm. to the center of the first spot. To the right and left of the sample at a distance of 2 cm, apply standard solutions containing 10, 5, 1 μg of the studied drugs (or other amounts close to the determined concentrations).

Plates with applied solutions are placed in a chamber for chromatography, on the bottom of which 30 minutes before the start chromatography, a mobile solvent is poured. When using records with a thin layer of alumina or silica gel, n-hexane is used as a mobile solvent or a mixture of hexane and acetone in a ratio of 6: 1, for drugs, in which the R value in hexane is below 0.3. Using f plates "Silufol" mobile solvent - 1% solution of acetone in hexane, and Silufol plates impregnated with o-tolidine - hexane with diethyl ether in a ratio of 49:1. The edge of the plate with applied solutions can be immersed in a mobile solvent no more than 0.5 cm.

After the solvent front rises by 10 cm, the plate is removed from the chamber and left for several minutes to evaporate the solvent. Next, the plate is irrigated with a developing reagent and exposed to UV light for 10 - 15 minutes (PRK-4 lamp). The plates should be placed at a distance of 20 cm from the light source.

In the presence of organochlorine pesticides, gray-black spots appear on the plate. When using Silufol plates impregnated with o-tolidine for analysis, they are immediately after chromatography subjected to UV irradiation for several minutes. In the presence of organochlorine pesticides, blue spots appear in this case. Quantitative determination is carried out by comparing the areas of the spots of the sample and standard solutions. There is a direct proportional relationship between the amount of the drug in the sample, not exceeding 20 µg, and the area of its spot on the plate. With a higher content of the drug, a proportional part of the studied extract should be used.

Chapter 4. MODERN HARDWARE DESIGN

SYSTEM FOR THIN-LAYER CHROMATOGRAPHY WITH DENSITOMETER "DenScan"

Purpose and scope

Systems for thin layer chromatography and electrophoresis with a DenScan densitometer are designed for qualitative and quantitative analysis of the composition of samples of substances and materials in the visible region of the spectrum and ultraviolet light at wavelengths of 254 and 365 nm.

Scope - research in chemistry, biochemistry, biology, medicine, pharmacology, analytical control of pure substances, environmental objects, etc.

Technical details

Densitometer provides calculation of parameters and quantitative evaluation of chromatograms in the visible and ultraviolet regions of the spectrum (lmax = 254 nm, lmax = 365 nm)

· The size of the processed plates, cm ............................................. no more than 15 x 15

· Image input time, s ............................................. .......... no more than 5

Chromatogram measurement time, min.................................... ………5

Signal-to-noise ratio: visible area .............. not less than 5/1

· UV, 254 nm.............................................. ......................... at least 5/1

· UV, 365 nm.............................................. ................ at least 5/1

· Relative RMS by spot area, %

· visible area .................................................................. ................ no more than 5

· UV, 254 nm.............................................. ......................... no more than 5

· UV, 365 nm.............................................. ......................... no more than 5

Range of Rf values: visible area .......... no more than 0.02

· UV, 254 nm.............................................. ................ no more than 0.02

· UV, 365 nm.............................................. ................. no more than 0.02

Mass of the lighting chamber, kg ............................................. no more than 12 kg

· Overall dimensions of the lighting chamber, mm.... no more length................................................. ............................... 420

width................................................. ............................... 420

height................................................. ............................. 700

· Supply voltage, V .............................................. 220 ± 22/33

· Frequency of alternating current, Hz .............................................. 50±1

· Mean time between failures of the densitometer, h.... not less than 5000

The composition of the densitometer

The "DenScan" densitometer consists of an illumination camera, a black-and-white or color video camera or a scanner, an image input unit, and a data processing system.

The lighting chamber is made in the form of a block structure, including the following main nodes:

Sources of light:

daylight lamps

UV lamps, wavelength 254 nm

UV lamps, wavelength 365 nm

A set of corrective filters

The detector is a black-and-white small-sized video camera OS-45D or similar with a sensitivity of at least 0.02 lux, with manual focus and manual adjustment of the aperture, or a color scanner with a resolution of 200 d.p.i. and above with an interface that complies with the TWAIN standard

Setting table for inserts

Communication channel with image input block

Data processing system using a personal computer and "Dens" software. Minimum computer requirements:

Operating system - Microsoft Windows 95, Windows 98, Windows NT (version 4.0 or higher)

Processor - Pentium 100 MHz

Color monitor - with a diagonal of at least 14 inches

Hard disk space - 10 MB

Manipulator - "mouse"

Image input block video blaster AverMedia ( and software for it) is used to obtain an image of the chromatogram on a computer monitor. It is possible to use similar systems.

Plates and sheets for thin layer chromatography (TLC)

Syringe for chromatography МШ-50 (М-50) Syringe for chromatography M-1N (MSh-1), M-5N (with guide)

Syringe for chromatography MSH-10 (M-10N), MSH-50 (M-50N) (stainless steel stem, with guide)

Syringe for chromatography МШ-10М (М-10) (stainless steel stem, with anti-recoil clutch) 10

Literature

1. Kirchner Yu. Thin layer chromatography. M.: Mir, 1981.

2. Chromatography in thin layers, Ed. E. Stahl. M.: Mir, 1965.

3. Evgen'ev M.I., Evgen'eva I.I., Moscow N.A., Levinson F.S. 5-Chloro-4,6-dinitrobenzofurazan as a reagent in thin-layer chromatography of aromatic amines // Zavod. lab. 1992. V. 58, No. 4. S. 11-13.

4. Nazarkina S.G. Determination of polyaromatic hydrocarbons in environmental objects by liquid and thin layer chromatography.

5. Sogolovsky B.M. Sorbfil densitometer for quantitative TLC

6. Guidelines for the chemical analysis of land surface waters (edited by A.D. Semenov) // Leningrad: Gidrometeoizdat. - 1977. - 540 p.

7. Unified methods of water analysis. Edited by Yu.Yu. Lurie // M.: Chemistry. - 1973. - 376 p.

8. Lurie Yu.Yu. Analytical chemistry of industrial and waste waters. // M.: Chemistry. - 1984. - 447 p.

9. V.D. Chmil Status and prospects for the use of modern instrumental methods for the analysis of pesticides in Ukraine

10. http://www.izme.ru/

Reversed-phase HPLC (RP HPLC) has a number of advantages over other liquid chromatography options:

– this is a very flexible method, since, by changing the composition of water-organic mixtures used as a mobile phase, it is possible to separate compounds of various nature on one column;

– the selectivity of this method is almost always significantly higher than other chromatography options for all compounds except highly polar

– when using hydrophobized silica gels, equilibrium between the mobile and stationary phases is quickly established; these sorbents are distinguished by high separation efficiency;

– it is possible to carry out the separation of compounds soluble both in water and in organic solvents;

– the possibility of using buffer solutions in the mobile phase can improve the selectivity and efficiency of separation of ionogenic compounds.

In reverse-phase chromatography, the stationary phase is hydrophobized silica gels, which are obtained by treating silica gel with chloro- and alkoxysilane. Hydrophobized silica gels with grafted octadecyl groups (C18) are widely used in analytical practice. Grafting density is 1.1-2.3 nm-2.

AT Depending on the processing method, the properties of hydrophobized silica gels can change, so the properties of commercial columns from different companies are somewhat different. The carbon content is 5-20%. The degree of coverage of the silica gel surface with an organic modifier is 10-60%, in the best cases it reaches 90%. The presence of residual silanol groups leads to the fact that

adsorption and ion-exchange retention mechanisms always accompany reversed-phase. To reduce the number of silanol groups, the sorbents are additionally treated with trimethylchlorosilane (this is called endcapping). In table. 12 shows typical reverse phase sorbents. The most popular are silica gels of the following brands: bondopak, lichrosorb, porasil, separon, spherisorb, nucleosil, kromasil. The disadvantages of reverse-phase sorbents based on silica gel are the limited allowable pH range and the sorption activity of silanol groups. This shortcoming is largely devoid of the new generation columns of the Phenominex company, its Luna C18 column is stable in the pH range of 1.5-10.

Separation mechanism compounds in this variant of chromatography is still not completely clear. The most successful and widespread are the theory using Hildebrant's concept of solubility parameters and the solvophobic theory of Horvath-Melander. According to the theory based on the Hildebrant solubility parameters, the retention is determined by the molecular interactions of the separated substances with the mobile and stationary phases. The dependence of the capacity factor of a substance on the composition of the mobile phase is described by the equation

lnk = Aφ2 + Bφ + C (12),

where φ is the volume fraction of the organic component (modifier) in the mobile phase, A, B and C are constants.

However, the behavior of complex compounds with several functional groups often cannot be described by this dependence. More adequately, the patterns of retention of sorbates in RP HPLC are described by the solvophobic theory. Horwarth and Milander were the first to show that aqueous eluents containing no

Table 12. Sorbents for reverse phase HPLC

	Sp , m2 /g		Particle shape
		particles, µm

Adsorbsil C8			Irregular
Adsorbsil C18			Irregular
Adsorbsphere C8			spherical
Adsorbsphere C18			spherical
Altima C8			spherical
Altima C18			spherical
AlfaBond S8			Irregular
AlfaBond S18			Irregular
M-Bondopak S18			Irregular
M-Bondopak Phenyl			Irregular
Hypersil C8			spherical
Hypersil ODS			spherical
Zorbax S8			spherical
Zorbax ODS			spherical
Diasorb-130-C1			Irregular
Diasfer 130-C8			spherical
Diasfer-130-S18T			spherical
Lichrosorb RP-2			Irregular
Lichrosorb RP 18			spherical
			spherical
			spherical
Nucleosil C18			spherical
Partisil ODS-3			Irregular
Separon C18			spherical
Silasorb C2			Irregular
Silasorb C8			Irregular
Silasorb C18			Irregular
			spherical
Sferisorb C18

organic solvents could be used to separate polar biological molecules on octadecyl silica gel. Even in the absence of an organic component in the eluent, the interaction between the solute and the grafted hydrocarbon radicals

stationary phase, was the reason for the retention of the dissolved substance. This led to the conclusion that the retention in the reversed-phase variant is mainly determined by hydrophobic interactions.

The most important role in understanding the retention mechanism of reverse phase chromatography was played by the work of Horvath and his school. The essence of Horvath's theory is as follows. There is a fundamental difference between the processes of sorption on polar surfaces from relatively nonpolar solvents (“normal phase mode”) and sorption from water or highly polar solvents on nonpolar surfaces (“reversed phase mode”). In the first case, associates are formed between the molecules of sorbates and stationary phases due to Coulomb interactions or hydrogen bonds. In the second case, the association on the surface is caused by the so-called solvophobic interactions in the mobile phase. Polar mobile phases, especially those containing water, are characterized by a strong Coulomb interaction and the formation of hydrogen bonds between solvent molecules. All molecules in such solvents are bound quite strongly by intermolecular forces. In order to place a sorbate molecule in this medium, it is necessary to form a "cavity" between the solvent molecules. The energy costs for the formation of such a “cavity” are only partially covered by the interaction of polar groups in the sorbate molecule with polar solvent molecules. Non-polar molecules of the stationary phase are in a similar position with respect to the solvent. From the energy point of view, such a position is more favorable when the interface between the polar medium (solvent) and nonpolar fragments of the stationary phase and sorbate molecules is minimal. The reduction of this surface is achieved during sorption (Fig. 15).

Rice. 15. To the mechanism of reversed-phase chromatography: a - sorbate in solution; b - sorbate on the surface of the stationary phase. Water and organic solvent molecules are indicated by light and dark circles, respectively.

Reverse phase chromatography is widely used not only to separate neutral compounds, but also ionic substances. In principle, the sorption process for such compounds is also described by the solvophobic theory. However, sorbates of this kind exist in solution and in the adsorbed state, both in the form of neutral molecules and in the form of ions. Each of these forms has its own retention factor value. Depending on the pH of the medium, the ratio of various forms in solution and the retention factors change.

Mixtures of solvents are usually used as the mobile phase, because this improves the selectivity and separation efficiency and reduces the time required for its implementation.

By changing the composition of the mobile phase in RPLC, the retention can be changed over a very wide range. For almost all analyzed compounds, the retention in some pure solvents (methanol, tetrahydrofuran) is negligible, while in pure water it is extremely high. Therefore, in order to achieve an acceptable retention time,

it is usually necessary to use mixtures of water with an organic solvent - the so-called modifier. The dependence of the substance retention factor on the composition of the mobile phase is described by the equation


where C is the concentration of organic	component (modifier) in

mobile phase, b and p are constants.

Under constant chromatographic conditions, the retention of various sorbates is determined by the following factors:

– hydrophobicity of sorbates;

– dipole moment;

– the volume of their molecules;

– polarizability;

– a decrease in the non-polar surface area during sorption.

When describing the relationship between retention and properties of sorbates, the most popular equations relate retention factors measured in a chromatographic system with distribution coefficients (most often in the octanol–water system). For compounds with similar structures, a linear relationship is observed between the logarithms of the coefficients

where Pi,j is the distribution coefficient of the substance between the aqueous and organic phases.

In many cases, the logarithm of the retention factor is linearly related to

The most common descriptor is the number of carbon atoms. These ratios are useful both in selecting the composition of the mobile phase

both in separation and to identify the components of a mixture.

To solve each specific problem, the composition of both the mobile and stationary phases must be carefully selected from the point of view of both the physical and chemical properties of its components. The general scheme for selecting the HPLC variant depending on the nature of the substances to be separated is shown in Fig. 3. 16.

HPLC Separation System consists of several blocks: a pump, a dispenser, a column, a detector and a recording device.

Consider the main types of pumps used in HPLC.

syringe pumps. The rotation of the precision synchronous motor is converted into the movement of a piston in the cylinder. When the piston moves, the mobile phase either enters the cylinder or is squeezed out of it. The advantage of this type of pump is the almost complete absence of pulsations in the flow of the mobile phase, the disadvantage is the impossibility of creating a gradient using a single pump.

Air booster pumps. Provide constant pressure at the inlet to the column. Advantages – no flow pulsations, high reliability; the disadvantage is the low reproducibility of the volumetric supply of the mobile phase.

Plunger reciprocating pumps. With the help of an electromechanical device, it is driven toreciprocatingthe movement of a plunger moving in the working head, as a result of which the pump either picks up the mobile phase or delivers it at a given speed. The advantage is a constant volumetric supply of the mobile phase, the disadvantage is rather large flow pulsations, which are the main cause of increased noise and a decrease in the sensitivity of the detector.

Rice. 16. Selection of HPLC conditions taking into account the hydrophobicity of the substances to be separated

To introduce a sample in liquid chromatography, the following types of dispensers are used:

– dosing loop

– Membrane dispensers (without flow stop and with flow stop)

Main types of detectors and their characteristics are given in table. 13. The most common detector in adsorption HPLC is spectrophotometric. In the process of elution of substances in a specially designed microcell, the optical density of the eluate is measured at a preselected wavelength corresponding to the absorption maximum of the substances being determined. Such detectors measure the absorption of light in the ultraviolet or visible region of the spectrum, with the former being used more frequently. This is due to the fact that most chemical compounds have fairly intense absorption bands in the wavelength range of 200-360 nm. Photometric detectors have a fairly high sensitivity. The sensitivity of the UV detector can reach 0.001 units. optical density per scale at 1% noise. With such a high sensitivity, up to several ng of even weakly absorbing UV substances can be detected. The wide range of linearity of the detector makes it possible to analyze both impurities and the main components of a mixture on a single chromatogram. The capabilities of the spectrophotometric detector have been significantly expanded after the advent of its modern analogue, the diode array detector (DMA), which operates in both the UV and visible regions. In such a detector, the "matrix" of photodiodes (there are more than 200 of them) constantly registers the absorption of electromagnetic radiation in the scanning mode. This makes it possible to record, at high sensitivity, undistorted spectra of rapidly passing through

component detector cell. Compared to detection at a single wavelength, comparison of the spectra obtained during peak elution allows the identification of separated components with a much greater degree of certainty.

Operating principle fluorimetric detector based on the measurement of fluorescent emission of absorbed light. Absorption is usually carried out in UV areas spectrum, the wavelengths of fluorescent radiation exceed the wavelengths of absorbed light. Fluorometric detectors have very high sensitivity and selectivity. Their most important area of application is the detection of aromatic polycyclic hydrocarbons.

Amperometric detector used to determine organic compounds that can be oxidized on the surface of a solid electrode. The analytical signal is the value of the oxidation current. The detector has at least two electrodes - a working electrode and a reference electrode (silver chloride or steel), sometimes an auxiliary electrode is installed, which is necessary to suppress the effect of an ohmic voltage drop in solutions of low conductivity. The success of the determination determines the choice of material and potential of the working electrode. The amperometric detector uses electrodes made of carbon materials, most often glassy carbon, and metal: platinum, gold, copper, nickel. The potential of the working electrode is set in the range of 0 - +1.3 V. Measurements can be carried out either at a constant potential or in a pulsed mode, when a three-stage potential sweep is set, which provides at different stages - oxidation of the substance, cleaning of the electrode and its regeneration. Using this

The detector is especially important when determining phenols, phenolic compounds, hydrazines, biogenic amines and some amino acids.

Conductivity detector used to determine inorganic anions and cations in ion chromatography. The principle of its operation is based on measuring the electrical conductivity of the mobile phase during the elution of a substance.

Table 13. High Performance Liquid Chromatography Detectors Used in Environmental Analysis

Type of detector	measured	Minimum	Selectivity
	parameter	defined
		quantity, g

Spectrophoto-	Optical	10 -10
metric	density
Fluorimetry	Intensity	10 -11
	fluorescence
Conductivity-	Electric wire-	10-9
ric
Amperometric	Current value	10-11 - 10-9
Amperometric	Current value	10-11 - 10-9

Mass Spectro-	the value	10 -12 – 10 -10
metric	ion current

Exceptionally informative is the mass

spectrometric detector , which has high sensitivity and selectivity. The main problem hindering the use of this detector is the problem of introducing the eluent flow into the mass spectrometer. The development of microcolumn chromatography allows

develop systems for direct injection of the eluent stream into the ion source of the mass spectrometer. Use high resolution mass spectrometers

and sufficient speed with chemical ionization at

atmospheric pressure or electrospray ionization. The latest models of mass spectrometers for liquid chromatography operate in the mass m/z range from 20 to

4000 amu The mass spectrometric detector imposes stringent requirements on the purity of solvents, is expensive and complex.

in circulation.

3.1.2. Using Reversed-Phase High Performance Liquid Chromatography to Solve Environmental Problems

Determination of water and soil pollution. High performance liquid chromatography is actively used to determine various ecotoxicants in waters and soils. The most significant tasks solved by HPLC in the analysis of water and soil are the determination of phenolic compounds, PAHs, and pesticides. Since the MPCs of these ecotoxicants in waters and soils are very low, their determination is usually carried out after preliminary concentration or isolation. Liquid extraction can be used for this, but sorption or solid phase extraction is a more convenient and efficient method.

Determination of phenols in waste and natural waters. Very common ecotoxicants are phenol and its chlorine and nitro derivatives, guaiacol, and cresols. These compounds are formed in the process of human production activities, in particular, inpulp and paperproduction. There is a need to determine them in various types of waters: natural,

plumbing, industrial and waste. The composition of waters is very complex and may include a large number of phenolic compounds, which are formed both at the stage of pollution and in the process of water purification. The most likely wastewater components are phenol, guaiacol, o-, m- and p-cresols, mono-, di-, tri- and pentachlorophenols, mono- and dinitrophenols. For the separation and simultaneous determination of volatile and low-volatile phenols, it is very successful to use high-performance liquid chromatography on hydrophobized silica gel. The efficiency and selectivity of the separation of phenols is determined by the composition of the mobile phase. Most often, mixtures of acetonitrile or methanol with buffer solutions (acetate or phosphate) are used to separate phenols in HPLC; successful separation of phenols of various compositions can be achieved if water acidified with acetic, chloroacetic, or phosphoric acid is used as the aqueous component of the mobile phase. The retention time of phenols is determined by their hydrophobicity and increases with its growth. For the most significant phenols, environmental pollutants, retention increases in the series: catechol< фенол < 4-нитрофенол < гваякол < п-крезол < 2,4-нитрофенол < 2-нитрофенол < 2-хлорфенол < 4- хлорфенол < 3-хлорфенол < 2,4-диметилфенол < 4-хлор-3-метилфенол < 2,4-дихлорфенол < 2,4,6- трихлорфенол < пентахлорфенол и зависит от состава подвижной фазы. Чем больше в ней содержание ацетонитрила или метанола, тем меньше удерживание. Для разделения столь сложной смеси фенольных соединений не удается подобрать подвижной фазы определенного состава. Необходимо либо использование градиентного элюирования, либо разные фенолы делят с использованием различных подвижных фаз.

Low MPCs of phenolic compounds in waters require sensitive detection methods or preliminary

concentration. The detection of phenols using DDM is quite successful; the limit of detection of phenol at a wavelength of 260 nm in this case reaches 1 mg/l. An amperometric detector has even greater sensitivity and selectivity to phenol and its derivatives. Its use makes it possible to determine phenols at the MPC level even in natural waters. In natural waters, MPC for phenol is 0.001 mg/l, p-chlorophenol - 0.002 mg/l, 2,4-dichlorophenol - 0.004 mg/ml, 2.4.6 - trichlorophenol - 0.006 mg/l and pentachlorophenol - 0.01 mg/l. Amperometric detection is based on the oxidation of phenols on the surface of a solid electrode, which is usually a glassy carbon electrode. It has been established that the maximum signal is recorded at the potential of the glassy carbon electrode – +1300 mV relative to the steel electrode or +1100 mV relative to the silver chloride reference electrode. It is important to use phosphoric acid as a component of the mobile phase; in this case, the fluctuations of the baseline of the amperometric detector signal are minimal, which makes it possible to reduce the value of the minimum detectable concentration, which corresponds to a signal equal to twice the “width” of the baseline. In table. 14. examples of the determination of phenol in waters under various conditions are given, in fig. 17 shows the chromatogram of the mixture, and fig. 18 - 20 determination of phenols in tap and waste water.

Definition of pesticides. In modern agriculture, chemical compounds used to combat pests, fungi, weeds, the so-called pesticides, are widely used. Along with the undoubted benefits, large-scale production and uncontrolled use of pesticides has led to a significant aggravation of the environmental situation.

Table. 14. Examples of determination of phenolic compounds in HPLC waters

Determined phenols	Stationary phase	mobile phase	Detector	сmin, mg/l
Catechol, phenol, 4-nitrophenol, 2-	Spherisorb C18,	Methanol (MeOH) - 1%		0.03 ─0.1(straight
nitrophenol, p-cresol, 2,4-dinitrophenol,		acetic solution
2,4-dimethylphenol, 2-chlorophenol, 4-		acid gradient		(0,65 ─ 1,0) 102
chlorophenol, 2,4-dichlorophenol, 2,4,6-				(preliminary
trichlorophenol, pentachlorophenol		25 ─ 100% MeOH		concentration
	Hypersil Green C18
	Hypersil Green C18	Acetonitrile (AN) - 1%		(0,3 – 8,0) 102
		Acetonitrile (AN) - 1%		(0,3 – 8,0) 102
		acetic solution		(preliminary
		acids; gradient		concentration

	Kromasil C18 5	30 ─ 100% AN		(2,5 – 27) 103
	Kromasil C18 5
		MeOH - H2 O;		(0,04 – 0,3) 103
		gradient mode:		(0,04 – 0,3) 103
Phenol, 2-chlorophenol, 2,4-dichlorophenol, 2,4,6-		25 ─ 100% MeOH
Phenol, 2-chlorophenol, 2,4-dichlorophenol, 2,4,6-
trichlorophenol, pentachlorophenol		AN ─ 0.1% H3 PO4 solution
Phenol, guaiacol, p-cresol, o-cresol,
Phenol, guaiacol, p-cresol, o-cresol,		AN ─ 0.1% H3 PO4 solution
		AN ─ 0.1% H3 PO4 solution
Pyragallol, 4-hydroxyaniline, benzcatechol,
Pyragallol, 4-hydroxyaniline, benzcatechol,
2-hydroxyaniline, phenol, cresols, mono-,	Silica gel C18,	MeOH ─ 0.1 M solution		8 10-5 – 4 10-4
di-, trichlorophenols, mono-, dinitrophenols,		Na2 HPO4 ─ 50 nM	cells	8 10-5 – 4 10-4
pentachlorophenol		nitrile triacetic
		acid ─ 0.03 M solution
		sodium dodecyl sulfate;
		gradient mode

Rice. 17. Chromatogram of the mixture: 2 - phenol; 3 - guaiacol; 4 - p-cresol; 5 - o-cresol; 6 - chlorocresol; 7 – p-chlorophenol; 1 - system peak. Column: (150x4.6) mm, Mightysil RP-18; Mobile phase:

acetonitrile:water:phosphoric acid (20.0:79.9:0.1)% v/v

Rice. Fig. 18. Chromatogram of a wastewater sample from a pulp and paper mill: 1 – systemic peak; 2 - 2,4,6-trichlorophenol; 5 - pentachlorophenol; 3,4,6 - unidentified peaks.

Column (150x4.6) mm Mightysil RP-18; Mobile phase:

acetonitrile:water:phosphoric acid (70.0:29.9:0.1) %v/v Mobile phase feed rate 0.7 ml/min. Amperometric detector. Working electrode potential 1300 mV

Rice. Fig. 19. Chromatogram of tap water with the addition of phenols (1 µg/l) with preliminary ion-pair extraction: 1 – phenol; 2 – 4-nitrophenol; 3 - 2,4-dinitrophenol; 4 - 2-chlorophenol; 5 - 2-nitrophenol; 6

– 2,6-dimethylphenol; 7 - 2,4-dimethylphenol; 8 – 2-methyl-4,6-dinitrophenol; 9 – 4-chloro-3-methylphenol; 10 - 2,4-dichlorophenol; 11-2,4,6-trimethylphenol; 12 - 2,4,6-trichlorophenol; 13 - pentachlorophenol. Column: steel (250x4.6 mm), Spherisorb ODS-2, 5 µm; Mobile phase: methanol - 1% acetic acid, gradient mode (methanol 25-100%); spectrophotometric detector, 280 nm (pentachlorophenol 302 nm)

Rice. Fig. 20. Chromatogram of tap water sample with phenol additives: 1 – phenol (0.1 µg/l); 2 - 2-chlorophenol (0.1 µg/l); 3 - 2,6-dichlorophenol (0.2 µg/l); 4 - 2,4-dichlorophenol (0.2 µg/l).

Phenols were concentrated from 30 ml.

Column (150x4.6) mm Mightysil RP-18. Mobile phase:

acetonitrile:water:phosphoric acid (70.0:29.9:0.1) %v/v The flow rate of the mobile phase is 0.7 ml/min. Amperometric detector; potential of the working electrode - 1300 mV

Since pesticides enter the body of people who do not have professional contact with pesticides, mainly with food and water, a permanent system for analyzing the quality of agricultural products, food and water is needed. At the same time, methods of analysis that could be used not only in scientific research but also in large-scale serial analytical control are of the greatest interest. Given the high toxicity of pesticides, monitoring requires specific and very sensitive analytical methods that allow the determination of pesticide residues and their metabolites at the trace level.

Chromatographic methods of analysis are more sensitive and make it possible to distinguish between related compounds and their metabolites or hydrolysis products. Recently, HPLC has been increasingly used for the determination and separation of pesticides. The method is most useful when analyzing low volatile or thermally unstable pesticides that cannot be analyzed using gas chromatography.

HPLC is most successfully used for the determination of carbamates, ureas, herbicides based on phenoxyacetic acids, triazines and their metabolites, benzimidazoles, and some other compounds.

One of the most popular herbicides are triazines, most of which are derivatives of s-triazine, a six-membered heterocycle with symmetrically arranged nitrogen atoms. Substituents are located in position 2,4 and 6. Three triazines are the best known: propazine, atrazine and simazine, the last two are included in the list of priority pollutants for EU countries. The maximum allowable concentration of triazines in drinking water is set at 100 ng/l. When analyzing waters, triazines are usually preconcentrated and then separated by RP HPLC. The stationary phase is hydrophobized silica gels, the mobile phase is a mixture of acetonitrile with water or buffer solutions. Triazines are detected using a diode array detector, UV, amperometric and mass spectrometric detectors. Examples of the determination of triazines by HPLC in water and soil are given in Table. fifteen.

Table 15. Examples of determination of pesticides in water and soil by HPLC

Detected pesticides	Stationary phase	mobile phase	Detector	Сmin, mg/l
Triazines: atrazine, simazine, propazine,	Ultracarb C18,	Acetonitrile (AN) - 1mM		preliminary
promethine, tetbutylazine, deethylatrazine,		phosphate buffer		concentration
deisopropylatrazine, hydroxyatrazine		solution, pH 7		(0.8-3.0)10-3 mg/kg
		gradient mode
		15 - 70% AN
Triazines: hydroxyatrazine,	Hypersil C18	Acetonitrile (AN) - 1mM	ampere	2.10-5 M
hydroxysimazine, hydroxydeethylatrazine		phosphate buffer	ric
		solution, pH 6.5
		gradient mode
		30–100% AN
Phenylurea derivatives:	Supelkosil C18 ,	AN– H2 O		preliminary
Monuron, flumetiron, Diuron, siduron,		gradient mode		concentration
linuron, neburon		40 - 90% AN		(2-4)10-3
				(0,4-3)10-4
Sulfonylureas
Chlorsulfuron, methylsulfuron,	Ultraspher C18,	MeOH–H2 O (pH 2.5),		preliminary
chlorimuron, thifensulfuron		gradient mode		concentration
	Viospher C6, 5 µm	40–70% MeOH
Cinosulfuron, tifensulfuron, methyl-	LiChrospher C18,	MeOH - 0.1% H3 PO4		0.01-0.05 mg/kg
sulfuron, sulphometuron, chlorsulfuron
Carbamates: carbaryl, profarm, methiocarb,	Supelkosil C18 ,	AN– H2 O (55:45)		preliminary
promecarb, chlorprofam, barban				concentration
				(0,3-8)10-3

7. Quaternary ammonium salts: paraquat, diquat, difenzoquat, chlormequat chloride, mepiquat

8. Acid herbicides: dicamba, bentazone, benazoline, 2.4 D, MCPA(2-methyl-4-chlorophenoxyacetic acid)

9. Derivatives of phosphonic and amino acids: glyphosate, glufosinate, bialofos

10. Mixtures of pesticides of various classes Simazin, fensulfothion, isoprocarb, fenobucarb, chlortilonil, etridiazol, mepronil, pronamide, mekrprom, bensulide, isofenofos, terbutol

11. Simazine, dichlorvos, thiram, 1,3-dichloropropene, fenobucarb, propizamine, iprofenfos, isoprothiolane, chlortilonil, fenitrothion, diazithione, isochathion, thiobencarb, chlornitrofen, azulan, iprodione, bensulin

12. Benomyl, 2,4-D, dicamba, rimsulfuron, chlorsulfuron, linuron, chlorsulfoxime, propiconazole, difenoconazole

			(0,1–10)10-4
			(0,1–10)10-4
Silica gel C18,	AN with NaCl additives,		4.4.10-4 mg/kg
	MeOH - solution
	hydroxide
	tetramethylammonium
LiChrosorb C18	MeOH - 0.01 M triethyl		preliminary
	amine, pH 6.9		concentration
	gradient mode		(0,2–1,0)10-4

	MeOH - 0.05 M NaH2PO4,
		Fluorescent	0,2.10-4
Nova-Pak C18	AH - 0.05M NaH2PO4,		(0,3–1.0)10-4
LiChrosorb NH2	0.02 M TMA bromide
LiChrosorb NH2
capillary	AN -H2 O		preliminary
LC column	gradient mode		concentration
Parkings C18,			(0,15–0,8)10-3

	AN - 1mM phosphate		preliminary
	buffer solution, pH 6,		concentration
	gradient mode		(0,04–0,5)10-3

Diaspher C16, 5 µm	AN - 0.01 M phosphate
	buffer solution, pH 4.2

Another group of pesticides for which HPLC is more promising than capillary gas chromatography are phenylurea derivatives. The most famous of them are linuron, monolinuron, pyrazon, and sulfonylureas (chlorsulfuron, tifensulfuron, rimsulfuron, methylsulfuron, etc.).

HPLC is also widely used for the separation and determination of carbamates. Particular attention is paid to the definition of carbaryl, propharma, methiocarb. The conditions for the separation of phenylureas, sulfonylureas, and carbamates are close to those for the separation of triazines.

The range of detectors used includes: diode array detector, UV, fluorimetric and mass spectrometric detectors. The amperometric detector is widely used. This detector gives a gain in sensitivity compared to UV in the determination of carbamate and urea derivatives (aldicarb, carbaryl, chlorpropharma, dimethoate, methiocarb) by about 10 times. Some examples of the separation of sulfonylureas, phenylureas and carbamates are shown in table. 15 and in fig. 21.

Selective herbicides - derivatives of phenoxyacetic acid (2,4-D, dicamba, bentazon, trichlorpyr, etc.), it is also preferable to determine HPLC. Hydrophobic silica gels serve as the stationary phase, and mixtures of acetonitrile or methanol with buffer solutions or water with the addition of acids serve as the mobile phase. The choice of pH of the mobile phase is especially important in the analysis of acidic compounds; its value is chosen lower than the pKa of the compounds to be separated. An ion-pair version of reverse-phase HPLC can also be used to increase separation selectivity.

Rice. Fig. 21. Chromatogram of the soil extract with the addition (10 µg/g) of herbicides, phenylurea derivatives: 1 – cinosulfuron; 2 - thiophenesulfuron methyl; 3 - methylsulfuron methyl; 4 - sulfometuron methyl; 5 - chlorsulfuron.

Steel column (100x4.6 mm), silica gel C18, 3 µm. Mobile phase methanol - 0.1% phosphoric acid solution (45:55). Spectrophotometric detector, 226 nm

Triethylamine is used as an ion-pair reagent to increase the retention of dicamba, bentazone, benazoline, 2,4-D and MCPA (2-methyl-4-chlorophenoxyacetic acid) on octadecyl silica gel in the neutral pH region. Thus, acidic herbicides are determined in drinking and groundwater (Table 15). Detection is carried out with a UV detector, the lowest detection limits are obtained for a UV detector with a diode array.

An important task is also the separation of mixtures containing pesticides of various classes, since in environmental objects they

hydrophobized silica gels: polar compounds are eluted already at a low content of acetonitrile (20-30)% in the mobile phase, more hydrophobic at a higher content (up to 70%), therefore, a gradient elution mode is used to separate mixtures. Examples of the separation of pesticide mixtures are shown in fig. 22, 23.

Rice. 22. Chromatogram of water with the addition of pesticides (0.2 mg/l) after preliminary sorption concentration: 1 - disisopropylatrazine; 2 - metamitron; 3 - chlordiazone; 4 - diethylatrazine; 5 - crimidine; 6 - carbetamide; 7 - bromacil; 8 - simazine; 9 - cyanazine; 10 - diethylterbutylazine; 11 - carbutilate; 12 - metabenzthiazuron; 13 - chlorotoluron; 14 - atrazine; 15 - monolinuron; 16 - isoproturon; 17 - metazachlor; 18 - metaprotrin; 19 - dimefuron; 20 - sebutylazine; 21 - propazine; 22 - tetbutylazine; 23 - linuron; 24 - chlorchuron; 25 - prometrin; 26 - chlorpropharm; 27 - terbutrin; 28 - metolachlor; 29 - pencicuron; 30 - bifenox; 31 - perdimethalin.

Column: LiChroCART (250x4 mm), Superspher 100 RP-18, 5 µm; mobile phase acetonitrile - 1 mM ammonium acetate (gradient mode - acetonitrile 25–90%). Spectrophotometric detector, 220 nm

Rice. 23. Chromatogram of the separation of a mixture of pesticides: 1-metabolite benomyl (2 µg/ml); 2 – acetamiprid (4 μg/ml); 3 – lenacil (10 μg/ml); four

– dicamba (4mcg/ml); 5 - chlorsulfuron (5 µg/ml); 6 - thiram (5 µg/ml); 7 - chlorsulfoxime (8 µg/ml); 8 - penconazole (5 µg/ml); 9 - linuron (5 µg/ml); 10 - fludioxonil (5 µg/ml); 11-propiconazole (5 µg/ml); 12 - difenoconazole (5 µg/ml).

Conditions for chromatographic determination: column Diaspher C16 (150x4.6) mm with an average particle size of 5 µm; mobile phase acetonitrile-0.01 M phosphate buffer solution (pH 4.2) (40:60). Mobile phase rate 1 ml/min. Spectrophotometric detector (230 nm)

The separation of organochlorine pesticides by HPLC is still being studied. This is partly due to the lack of publicly available selective detection methods after separating them by means of reverse phase chromatography. The limit of detection of organochlorine pesticides (DDT type) and esters of phenoxycarboxylic acids by absorption at 254 nm is 1-15 and 15 µg, respectively.

As a method for the analysis of residues of organophosphorus pesticides, HPLC has not received due distribution. These compounds are detected by absorbance at 254 nm, cholinesterase inhibition and

polarographically. The applicability of phosphorus-sensitive detectors for the selective detection of organophosphorus compounds in HPLC is shown.

One of the important issues that determine the sensitivity of the determination of pesticides is the method of detection. Most studies are characterized by the use of the spectrophotometric method, but its use is limited by a number of factors: not all compounds absorb well, different compounds have different absorption spectra. Therefore, it is very difficult to choose the appropriate wavelength. There may be other compounds in the environment, in the presence of which the determination of pesticides will be difficult.

Recently, the possibilities of electrochemical detection (ECD) in liquid chromatography have been widely studied. In an attempt to increase the sensitivity of HPLC detection of organochlorine pesticides, Dolan and Sieber constructed an improved version of the Coulson Electrolytic Conductometric Detector (ECDC). This detector is characterized by high selectivity in the determination of organochlorine compounds, its linear range corresponds to a change in the concentration within five orders of magnitude, and the lower limit of detection of lindane is 5–50 ng. The applicability of EPDC in an analytical system has been demonstrated in the analysis of raw extracts of lettuce leaves and river water containing aldrin and dieldrin at concentrations less than 10-4%. The use in this case of a UV detector with a wavelength of 254 or 220 nm does not allow the determination of aldrin and dieldrin.

The limits of detection achieved with the help of voltammetric detectors, the relative simplicity of the device, and an acceptable cost make this method quite suitable for the analysis of trace amounts of organic substances. When using an ECD operating in

recovery mode, one of the significant problems is the recovery of oxygen dissolved in the eluent, the peak of which can interfere with the determination of the analyte. There are various ways to remove dissolved oxygen, however, at such low detectable concentrations of pesticides, it is not always possible to get rid of its trace amounts. In this regard, if possible, the determination of pesticides is carried out in the anode potential region.

In combination with the HPLC method, amperometric detection is most often used, in which the potential of the working electrode is kept constant and the current that occurs during the oxidation or reduction of electroactive molecules is measured as a function of time. The amperometric detector makes it possible to detect with high sensitivity a wide range of pesticides: thiram, triazines (simazine, atrazine, cyanazine, propazine and anilazine), carbamate pesticides (barban, baygon, benomyl, chlorpropham, landrin, mesurol, profam, sevin, aminocarb, carbendazim, desmedifam ), phenylurea pesticides (metobromuron and linuron). These compounds are determined in waters using an amperometric detector, in most cases the detection limits are lower than with a spectrophotometric detector. For example, the limit of detection for aminocarb and carbendazim is less than 1 µg/l, desmedifam and dichloran are less than 5 µg/l, metamitron 10 ng/l, chlortoluron and isoproturon 20 ng/l.

Determination of polycyclic aromatic hydrocarbons

(PAH). Quite often, liquid chromatography is used to determine PAHs in waters and soils. If it is necessary to simultaneously determine medium and low volatile aromatic hydrocarbons, reverse-phase high performance liquid chromatography is usually chosen.

Due to the unique properties and wide availability of octadecyl silica gel (ODS) reversed phases, most PAH studies have been performed on these phases. With a decrease in the chain length of the grafted hydrocarbon radical, the values of the capacitance coefficient rapidly decrease, which significantly complicates the analysis of multicomponent mixtures of PAHs. Thus, under identical conditions (composition of the mobile phase, eluent flow rate, temperature, column dimensions), the retention time of PAHs on a column with Nucleosil C18 is about twice as long as on a Nucleosil C8 column. It is believed that PAH molecules are retained on the nonpolar surface of alkyl silica gel due to van der Waals forces, and the bond strength increases with increasing side chain length.

Sorbents with grafted polar groups are also used to separate PAHs. The radicals of alkyl(aryl)alkanes used to modify the surface of sorbents contain one or more polar groups (-NH2, -NO2, -OH, -CN, etc.). The mechanism of PAH retention on sorbents with grafted polar groups is quite complex.

The interaction between the π-electronic system of the sample components and various structures of the polar surface is taken into account. Unsubstituted PAHs are eluted in ascending order of molecular weight. In the polar phase containing amino groups, the retention of PAHs increases with an increase in the number of aromatic nuclei in the molecule. In contrast to columns with hydrophobic silica gels, the presence of alkyl groups in PAH molecules on polar phases has little effect on the retention order, which makes it possible to use these phases for preliminary fractionation in the analysis of complex mixtures of PAHs.

In practice, the separation of PAHs is more often carried out on hydrophobic silica gels, since the separation selectivity is higher, the reproducibility of the results is better, and a longer service life of chromatographic columns is also observed.

In reverse-phase chromatography, for the separation of PAHs, water-alcohol mixtures (water-methanol) and water-acetonitrile mixtures are most often used as eluents. The relative retention times for individual PAHs are very different, so the gradient elution mode is more often used.

There are many options for detecting PAHs: amperometric, fluorescent, ultraviolet. The most commonly used fluorescence detection of PAHs. HPLC combined with a fluorescent detector is a selective and sensitive method for the determination of PAHs in natural samples. A diode array UV-VIS spectrophotometric detector is useful for the quantitative and qualitative analysis of PAHs in soil samples in the nanogram range, while a fluorescent detector is recommended for the analysis of PAHs in water samples in the picogram range.

The highest sensitivity of a fluorescent detector can only be obtained at optimal excitation and fluorescence wavelengths for individual PAHs. This is only possible by programming these wavelengths in time. After optimization of all individual parameters, the minimum detection limit for individual PAHs in drinking water reaches the level of 0.5 picograms.

Widely accepted EPA methodologies recommend that naphthalene, acenaphthylene, acenaphthene and fluorene be determined with an ultraviolet detector and that a fluorescent detector be used for all other PAHs. On fig. 24 shows the separation of a mixture of 16 priority PAHs.

Rice. Fig. 24. Chromatogram of a standard mixture of EPA polycyclic aromatic hydrocarbons: 1 - naphthalene; 2 - acenaphthene; 3 - fluorene; 4 - phenanthrene; 5 - anthracene; 6 - fluoranthene; 7 - pyrene; 8 – 3,4-dibenzaanthracene; 9 - chrysene; 10 - 3,4-benzfluoranthene; 11 - 11,12-benzfluoranthet; 12 - 3,4-benzpyrene; 13 - 1,2,5,6-dibenzanthrace and 1,12-benzperylene; 14 - 2,3-o-phenylenepyrene.

Column (150x4.6mm) Mightysil RP-18; mobile phase: (75:25)

acetonitrile-water: detector ─ fluorescent, programming mode by fluorescence wavelengths

Determination of PAHs in environmental objects, especially in waters

and soils is an important problem in practical analytical chemistry.

AT There are many works in the literature devoted to the determination of PAHs by HPLC in waters and soils. The data of these works are summarized in Table 1, respectively. 16 and 17.

Difficulties in the determination of PAHs by HPLC are associated with the need for preliminary purification of extracts and the fundamental difficulties in identifying related

chemical

structure

isomeric

connections.

Table 16. Determination of PAHs by HPLC in waters

	Defined PAHs	Stationary phase	mobile phase	Detector	Cmin , ng/l
Drinking	Fl, B(b)F, B(k)F, B(a)P,		Acetonitrile: water
Drinking	B(g,h,i)P, Ind(1,2,3-cd)P	(250x4.6) mm, 5 microns	gradient mode
	B(g,h,i)P, Ind(1,2,3-cd)P	(250x4.6) mm, 5 microns	gradient mode
polluted			Acetonitrile: water
		(100x8) mm, 5 µm	gradient mode
		Lichrospher RAS S-18	Acetonitrile: water
		(125×2) mm, 4 µm	gradient mode
		(125×2) mm, 4 µm	gradient mode
Surface			Methanol: water (85:15) with
Surface		(250x4.6) mm, 5 microns
		(250x4.6) mm, 5 microns
		Spherisorb S5 RAS	Acetonitrile: water (80:20)
		(150×4.6) mm, 5 µm	isocratic mode
		(150×4.6) mm, 5 µm	isocratic mode
	Fl, B(b)F, B(k)F, B(a)P,		Methanol: water (85:15)
	B(g,h,i)P, Ind(1,2,3-cd)P	(165×4.6) mm, 5 µm	isocratic mode
	B(g,h,i)P, Ind(1,2,3-cd)P	(165×4.6) mm, 5 µm	isocratic mode
Surface			Acetonitrile: water
		(250x4.6) mm, 5 microns	gradient mode
		(250x4.6) mm, 5 microns	gradient mode
	Fl, P, B(a)P		Acetonitrile: water
		(150×4) mm, 5 µm)	gradient mode
		(150×4) mm, 5 µm)	gradient mode
Natural		Lichrospher 100 RP-18	Acetonitrile: water (80:20)		0.5 ng/l (B(a)P)
		(125×4) mm, 5 µm	isocratic mode		0.5 ng/l (B(a)P)
		(125×4) mm, 5 µm	isocratic mode
	Fl, B(b)F, B(k)F, B(a)P,	SpherisorbODS-2	Acetonitrile: water (80:20)		~ 8 pg (B(a)P)
	B(g,h,i)P, Ind(1,2,3-cd)P	(300×4) mm, 5 µm	isocratic mode		~ 8 pg (B(a)P)
	B(g,h,i)P, Ind(1,2,3-cd)P	(300×4) mm, 5 µm	isocratic mode
Urban		Hypersil Green PAH	Acetonitrile: water
		(100×4.6) mm, 5 µm)	Acetonitrile: water
		(100×4.6) mm, 5 µm)	gradient mode
			gradient mode

Notes: Fl - fluorescent detector; Amp - amperometric detector;

TCAA, trichloroacetic acid; i-PrOH, isopropanol; 16 PAH - 16 PAH from EPA Standard Blend

Fl, fluoranthene; P - pyrene; B(b)F, benzo(b)fluoranthene; B(k)F, benzo(k)fluoranthene; B(g,h,i) – benzo(g,h,i)perylene;

Ind(1,2,3-cd)P, indeno(1,2,3-cd)pyrene;

SO - limit of detection

Table 17. Determination of PAHs by HPLC in soils

soil type	Defined	motionless	Movable	C min,

Sedimentary		C18 ((250×4.6)	Acetonitrile:
deposits
			gradient

Soil		C18 ((250×4.6)	Acetonitrile:
	B(k)F, B(a)P,
			gradient


Strongly			Acetonitrile:
dirty			water (80:20)
		ODS((243×4)	isocratic
			cue mode
		C18 ((250×4.6)	Acetonitrile:
dirty
			gradient

Sedimentary		C18 ((250×4.6)	Acetonitrile:
deposits
			gradient

In the analysis of river water samples, since they may contain admixtures of fluorescent compounds, at relative PAH retention times, it is proposed to use preliminary separation of PAH fractions by thin layer chromatography (TLC) and subsequent analysis of individual PAH fractions by reverse phase HPLC with a fluorescent detector.

In soils and complex natural mixtures of PAHs, it may be necessary to use the normal-phase HPLC method to determine specific PAH isomers. This method provides separation and concentration of isomers that are difficult to determine in general

fractions of PAHs due to low concentrations or due to the relatively low sensitivity and selectivity of fluorescent detection. A method for separating a natural extract of marine sediments on aminopropyl silica gel is described. This preliminary stage provides the production of fractions containing only isomeric PAHs and alkyl-substituted isomers. Fractions of isomeric PAHs are analyzed by reverse-phase HPLC with a fluorescent detector.

Thus, HPLC using fluorescent and ultraviolet detectors makes it possible to determine PAHs in various objects. The success of the analysis is determined both by the conditions of separation and detection, and by the competent preparation of the sample for analysis.

Determination of air pollution. To determine contaminants in air, HPLC is used less frequently than in water and soil. This method is indispensable for the determination of toxic macromolecular and high-boiling organic compounds in the air: these include dioxins, pesticides, polychlorobiphenyls, PAHs, phenols, aromatic amines and imines, azarenes (nitrogen-containing heterocyclic hydrocarbons) and their methyl derivatives. In all cases, pre-contaminating components are captured from the air in special concentrating tubes, and after extraction from the adsorbent phase, the resulting HPLC solution is analyzed.

The most important is the determination of PAHs in the air (maximum concentration limit for atmospheric air is 10-6 mg/m3, air of the working area - 1.5.10-4 mg/m3), the analysis of the concentrate is carried out in the same way as described for water and soil. Much attention is also paid to the determination of phenols and cresols. This task is important for residential premises, since building materials, coatings, and furniture can release phenols. They are caught when air is pumped through alkaline solutions or on special

Chromatographic methods continue to be the main tool in the analytical chemistry of pesticides. In terms of development rates, capillary gas chromatography (GC), high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC/MS, LC/MS) occupy the first places among them. Capillary GC has no alternative when developing methods for the determination of multiple pesticide residues. p>A number of pesticides used in Ukrainian agriculture cannot be subjected to direct gas chromatographic determination due to their low volatility or insufficient thermal stability. In order to make it possible to determine these compounds using GC, they are converted into various derivatives. Such an operation usually increases the volatility and reduces the adsorption of the chromatographed compounds on solid carriers, increases their thermal stability and improves separation. In some cases, this also achieves a significant increase in the detection sensitivity of the obtained derivatives. All this is the subject of reactive gas chromatography. For the first time in domestic studies, we have shown the effectiveness of using reactive gas chromatography in the analysis of pesticides by the example of determining residual amounts of herbicides - derivatives of phenoxyalkanecarboxylic acids (2,4-D, 2,4-DM) in food products. Since then, the method of reaction gas chromatography has been widely used in the laboratories of the Institute when conducting state tests of pesticides and carrying out state sanitary and hygienic examination. AAAAAAAAAAAAAAAAAAAAAAAAAAA

Listing the chromatographic methods that are used in the analysis of pesticide residues, one cannot fail to mention the thin layer chromatography (TLC) method, which was discovered in 1938 by Ukrainian scientists N.A. Izmailov and M.S. Schreiber. Semi-quantitative TLC is currently an inexpensive and efficient method for the separation, identification and semi-quantitation of pesticide residues. It was the semi-quantitative version of TLC that played a big role in the formation of the chemical analytical service of the Ministry of Health of Ukraine for monitoring the content of pesticide residues in food and environmental objects, when GC and HPLC methods were not yet available for wide use. This was largely due to the work carried out within the walls of the Institute. Currently, TLC in the analysis of pesticide residues is mainly used as an alternative analytical method to confirm the correct identification of pesticides obtained using GC and HPLC methods. TLC is also an indispensable tool in the analysis of pesticide residues, when it is necessary to check a very large number of food or environmental samples for the presence of pesticides. In such cases, a screening methodology is usually applied. All samples that give a "positive" reaction are further analyzed by some more specific instrumental method (GC, HPLC, GC/MS, LC/MS), while all negative screen results are accepted as final without any verification. The Institute has a set of equipment for quantitative TLC (KAMAG, Germany). Nevertheless, the prospects for further use of TLC in the analysis of pesticides should primarily be associated with a semi-quantitative version of this method. There is no alternative to this.

Each stage of the use of pesticides in world agricultural practice from the late 40s of the last century to the present can be characterized by its own chemical and analytical problems. However, one problem in the analysis of pesticide residues remains unchanged - the need to constantly reduce the limits of quantitative determination (limit of quantitafication, LOQ) of pesticides. Achieving very low limits of quantitative determination when using MVI is accompanied by a decrease in the level of reliability (identification reliability) of the analysis result. Often, in order to achieve very low limits of quantitation, it is necessary to use a complex multi-step purification procedure and a derivatization step so that highly selective and highly sensitive detectors (ECD, TID) can be used. However, this is inevitably accompanied by losses of the analyte during these operations, which leads to an increase in the analysis error. In addition, the variability of the composition of the analyzed matrix from sample to sample also contributes. In this regard, an analytical chemist cannot always satisfy the desire of a hygienist and toxicologist to have MVI with very low limits of quantitative determination due to the technical capabilities of the instruments used and the methodological limitations of the MVI being developed. When developing MVI, the analytical chemist should focus his efforts not only on achieving low limits of quantitative determination of the analyzed pesticides, but not lose sight of the more important aspects of the analysis of pesticide residues: reliability of identification and reproducibility of results. It is known that at present in Ukraine in some agricultural crops and food products the content of pesticides is not allowed (the so-called zero tolerances) or is at the level of the limit of detection (LOD), i.e. any detectable pesticide residues are considered unacceptable. In such cases, the reliability of identification of the pesticide is of paramount importance, and not the exact quantitative determination of its content, since the very fact of the detection of a pesticide is the basis for a ban on the use of agricultural raw materials or food products. In these cases, the use of a semi-quantitative TLC variant is fully justified, provided that a reliable identification of the pesticide being determined is achieved.

Introduction

Chapter 1. Existing methods for determining the content of pesticides in the analyzed objects (literature review)

1.1. Sample preparation using solid phase extraction 6

1.2. Methods for qualitative characterization of pesticides 16

1.3. Quantitative analysis of pesticides 20

Chapter 2. Technique and experimental conditions

2.1. Determination of Pesticide Partition Coefficients in Hexane/Acetonitrile System Using Gas-Liquid and Reverse Phase High Performance Liquid Chromatography 24

2.2. Determination of the degree of extraction of pesticides from model aqueous solutions using solid phase extraction 30

2.3. Determination of linear-logarithmic retention indices and relative optical densities of pesticides in reverse phase high performance liquid chromatography 32

2.4. Quantitative assessment of the content of pesticides in plant objects by the methods of an external standard and a standard additive 34

2.5. Determination of the content of pesticides in real plant objects.39

Chapter 3. Assessment of the degree of extraction of pesticides from model aqueous solutions under conditions of solid-phase extraction based on their distribution coefficients in the hexane/acetonitrile system and hydrophobicity parameters

3.1. Features of the use of reverse-phase high-performance liquid chromatography in the determination of the distribution coefficients of pesticides in the hexane/acetonitrile system 42

3.2. Estimation of hydrophobicity parameters of potential organophosphate pesticides by their retention indices in reverse-phase high-performance liquid chromatography 48

3.3. Evaluation of the relationship between the degree of extraction of pesticides from aqueous solutions during solid-phase extraction with their coefficients in octanol/water and hexane/acetonitrile systems 59

Chapter 4. Interpretation of the results of identification and quantification of pesticides in plant objects

4.1. Selection of optimal analytical parameters for the chromatographic characterization of pesticides 63

4.2. Comparison of external standard and standard additive methods for assessing the content of pesticides in plant objects 71

References 92

Applications 105

Introduction to work

The widespread use of chemical plant protection products puts the analysis of pesticides in agricultural products and environmental objects into a number of priority tasks of environmental analytical control. In this regard, as well as with the new requirements imposed by Rostekhregulirovanie on control methods, there is a need to improve old and develop new methods for determining microquantities of pesticides [using gas-liquid (GLC) and high-performance liquid (HPLC) - chromatography], which would combine simplicity of the determination procedure with maximum reliability of the results obtained. New approaches to the determination of trace amounts of ecotoxicants can help to successfully solve this problem.

The most important stages in the analysis of pesticides are: sample preparation and final interpretation of the data, including both qualitative and quantitative characterization of the analyzed compounds. Sample preparation for analysis usually consists of extraction, re-extraction and column purification. Solid phase extraction (SPE) is an alternative approach to its implementation. It combines a number of the above procedures into one, which saves time and reagents. However, to optimize the SPE process, some information about the target substances is required, in particular, about their distribution coefficients in heterophase solvent systems 1-octanol/water (log P) and hexane/acetonitrile (Kp). In the reference literature on pesticides, along with other physico-chemical characteristics, log P values of pesticides are given. However, the problem of their determination is still relevant due to the existing difficulties that arise in the process of determination. The main one is

the formation of slowly separating emulsions of both solvents in each other. This is reflected in the low interlaboratory reproducibility of pesticide log P values. Therefore, it is important to systematically characterize pesticides of various chemical groups, primarily by their distribution coefficients in octanol/water and hexane/acetonitrile systems, as well as retention indices in reverse phase high performance liquid chromatography [RI (HPLC)]. The latter can be used not only to identify the analyzed compounds, but also to assess their hydrophobicity parameters. The expansion of such a database of physicochemical characteristics of pesticides and the range of characterized compounds will help, on the one hand, to carry out a full sample preparation, and, on the other hand, to identify them. However, for an unambiguous and reliable qualitative characteristic, one of the available parameters is not enough. It is necessary to evaluate the information content of various combinations of analytical parameters of pesticides, which will allow solving the problem of their identification with maximum reliability.

The final stage of analysis after sample preparation and qualitative characterization of the analyzed compounds is the quantitative assessment of their contents in the studied samples. The existing methods of quantitative chromatographic analysis of pesticides (absolute calibration, internal standard method) cannot be called optimal. The method of absolute calibration in the presence of systematic errors in sample preparation (as a rule, due to losses of the substances of interest at different stages) without the introduction of correction factors leads to underestimated results, and the use of the internal standard method is limited to the search for the necessary standard compound and a preliminary additional, laborious procedure for special sample preparation to carry out the definition.

6 Thus, the purpose of this work was to improve existing and develop new methods for the determination of pesticides in plant objects. To solve this problem, it is necessary to optimize each of the main stages of pesticide analysis. The proposed optimization includes: the use of SPE at the stage of sample preparation, and during the final interpretation of the data, the choice of the most optimal combination of analytical parameters for the chromatographic identification of pesticides, as well as the choice and use of a method for their quantitative assessment, which allows minimizing the systematic errors of determinations.

Methods for the qualitative characterization of pesticides

Identification of pesticides (as well as any other organic substances) during chromatographic analysis (GLC and HPLC) is often carried out by retention parameters [absolute and relative retention times, retention indices (linear, logarithmic, linear-logarithmic)] on phases of different polarity (GLC ) or in various elution modes (HPLC). Carrying out a qualitative analysis of pesticides by absolute times is carried out under strictly specified conditions, on the same device using the necessary standard (reference) compounds. Less dependent on the specific conditions of the analysis are the relative retention times (retention times relative to any standard substance). They have significantly greater reproducibility under isothermal separation conditions (GLC) and isocratic elution conditions (HPLC). They can be used to compare data obtained in different stationary modes, on different instruments, in different laboratories. However, the nature of the stationary phases (GLC), the type of columns and the composition of the eluent (HPLC) must remain fixed. As a standard connection, it is recommended to choose a connection of the same class as the one being defined. If, however, the retention parameters (retention indices (RI)) are determined relative to two standards, one of which has a shorter and the other a longer retention time than the desired compound, then they will be characterized by even greater interlaboratory reproducibility than relative retention times. Retention indices can be presented in linear, logarithmic and linear-logarithmic form. Retention indices in logarithmic form are used in isothermal mode (GLC) or isocratic elution mode (HPLC). In the case of the analysis of complex mixtures under conditions of programmed column temperature change (GLC), linear retention indices are used. However, as shown in the best form of representation of the retention parameters under these conditions are linear-logarithmic retention indices. Their advantage lies in high reproducibility both in linear temperature programming and isothermal mode (GLC), as well as in various elution modes (isocratic, gradient) of the mobile phase in HPLC. Retention indices have found use not only in the analysis of pesticides, but also in other organic pollutants. However, the use of chromatographic retention parameters is associated with ambiguous estimates. This is due to the real possibility of their coincidence with the retention parameters of co-extractive substances usually present in the sample (co-extractive substances are compounds extracted from the matrix together with the analyte).

Another way to identify substances is based on the use of selective detectors. Gas chromatographic analysis of pesticides is carried out using three selective detectors - thermal ion and flame photometric detectors are used in the analysis of nitrogen-, phosphorus-, sulfur-containing compounds, and the electron capture detector is used in the analysis of halogen-containing substances. The use of alternative detectors is limited in that although some of them are registered with the required sensitivity. Analysis of pesticides under reverse phase HPLC conditions is carried out with practically one selective ultraviolet (UV) detector, the selectivity of which is controlled by the choice of fixed wavelengths. The use of diode arrays makes it possible to record absorption at several wavelengths, thereby providing a greater likelihood of qualitative characterization of pesticides.

One of the most reliable ways to identify ecotoxicants are hybrid methods based on the chromatographic separation of the analyzed substances and subsequent identification using spectral (mass, infrared, atomic emission) detectors. In this case, in addition to chromatograms with determinable retention parameters, the corresponding (mass, infrared, atomic emission) spectra of compounds are recorded. However, as noted in , "none of the known analytical methods can guarantee the reliable identification of any compounds." To this it should be added that the use of hybrid methods is limited by expensive hardware. The advantages and limitations of each of the methods used for the qualitative characterization of pesticides are illustrated in Table 1.2.

Determination of linear-logarithmic retention indices and relative optical densities of pesticides in reverse-phase high-performance liquid chromatography

We used pesticides, the list of which is presented in Table 2.1., as well as compounds (1-23) with the general structural formula RRP(=X)SR (Table 2.2.), synthesized at the Institute of Organoelement Compounds (Moscow), physicochemical properties which are characterized in . Separation of the compounds by reverse-phase HPLC was carried out on a Waters liquid chromatograph with a Nova-Pac Qg column (3.9 x 150 mm) and UV detection at wavelengths of 220 and 254 nm. A mixture of acetonitrile with water was used as the mobile phase, the eluent flow rate was 1 ml/min. The analysis was carried out in a gradient elution mode with an initial CH3CN concentration of 10% and a rate of its change of 1.5% per minute. The dead time of the system was determined by dosing a solution of potassium bromide (220 nm). The retention times were recorded using the Millennium software. To determine the RI values, a mixture of reference n-alkylphenyl ketones PhCOCnH2n+i (n = 1–3.5) was introduced into the samples. Linear-logarithmic retention indices [RI(HPLC)] were calculated using the program (QBasic) provided in the manual. To calculate the RI values (HPLC) of compounds having retention times shorter than those of the first reference component (acetophenone), the retention time extrapolation algorithm described in . To determine the relative optical densities Aotn.= A(254)/A(220), chromatograms were recorded in parallel at the two specified wavelengths, followed by calculation of the peak area ratios Aotn.= S(254)/S(220). Calculation of the parameters of the linear regression equation of the form: log Р = al +b, where / - retention indices of substances in reversed-phase HPLC, a, b - coefficients of the equation; were carried out using Origin for Windows software.

Estimates of log P values according to additive schemes (based on the log P increments of molecular fragments) were performed using the ACD and CS ChemDraw Ultra software. Peculiarities of quantitative assessment of the content of pesticides in plant objects [cucumbers (frozen), straw, ears, grain] were characterized by the example of three compounds: dimethoate, pirimicarb and malathion. Standard solutions of pesticides in acetone (chemically pure) with a concentration of 0.1 mg/ml (and 0.01 mg/ml for dimethoate) were prepared by diluting the initial stock solutions with a concentration of 1 mg/ml and applied as evenly as possible (1-2 .5 ml) into untreated (control) plant samples, followed by shaking and mixing for 5 minutes. The absence of detectable pesticides in control samples was confirmed experimentally using Sample preparation for further chromatographic analysis was carried out in two ways: with LE (cucumbers, straw, ears, grain) and using SPE (cucumbers). Sample preparation using liquid extraction. Preparation of samples containing dimethoate and malathion was carried out according to the method of group determination of organophosphorus pesticides. It included the extraction of pesticides from cucumber samples with 50% aqueous acetone (an ultrasonic bath was used to increase the extraction efficiency).

The obtained extracts were filtered through a paper filter. The filter cake was washed with 50% aqueous acetone. Re-extraction of pesticides from water-acetone solutions was carried out with dichloromethane (three times 30 ml). The dichloromethane solutions were dried by passing them through a layer of anhydrous sodium sulfate (analytical grade) and evaporated to dryness in a fume hood at room temperature in a stream of air. The dry residue was dissolved in 10 ml of hexane and chromatographed. The preparation of samples containing pirimicarb was carried out using the procedure given in. It is based on the extraction of the pesticide from the analyzed objects with a 0.1 N hydrochloric acid solution. The obtained extracts were alkalized with 1 N sodium hydroxide solution to pH 8–10 and pirimicarb was re-extracted with chloroform (two portions of 75 ml each). The chloroform extracts were dried by passing them through a layer of anhydrous sodium sulfate and evaporated to dryness in a fume hood at room temperature in an air stream. The dry residue was dissolved in 10 ml of hexane and chromatographed. Sample preparation using solid-phase extraction. Pesticides were extracted from the analyzed samples with 50% aqueous acetone (in an ultrasonic bath). After filtration of aqueous acetone solutions and washing of the filter cake (50% aqueous acetone), acetone from the combined extracts was completely evaporated. The remaining aqueous solutions were again filtered through filter paper. Before using the domestic sorbents Diapak C16 (batch No. 1002), they were activated (activation of the cartridges, see paragraph 2.2. above). After that, the analyzed aqueous solutions were pumped through the cartridges at a rate of not more than 2 ml/min, creating a vacuum at the outlet with a water jet pump. Then the cartridges were dried for 30 minutes in a helium flow. As eluting solvents were used: hexane (20 ml), dichloromethane (20 ml) and acetone (15 ml). The eluates were evaporated to dryness in a fume hood at room temperature.

The residues after evaporation were dissolved in 10 ml of hexane and chromatographed. Gas chromatographic analysis with the combined presence of dimethoate, pirimicarb and malathion was performed using the Tsvet 55OM instrument, equipped with a thermionic detector and a 2 m x 3 mm glass column filled with 5% SP 2100 on Chromosorb W (0.200 -0.250 mm). Column temperature 220, evaporator 250, detector 390C. Carrier gas (nitrogen) consumption - 30 ml/min, hydrogen 14 ml/min, air 200 ml/min. Gas chromatographic analysis of dimethoate was carried out on a Tsvet 550M instrument with a thermionic detector and a 1 m x 3 mm glass column filled with 5% SE-30 on Chromaton N Super (0.125 - 0.160 mm). Column temperature 200, evaporator 240, detector 320C. Carrier gas (nitrogen) consumption - 28 ml/min, hydrogen 14 ml/min, air 200 ml/min. Samples (1 µl) were dosed using a Hamilton microsyringe. The quantitative assessment of the content of pesticides in the analyzed samples using the external standard method was carried out according to the equation (in all cases, the analyzed volumes were the same and amounted to 10 ml):

Estimation of Hydrophobicity Parameters of Potential Organophosphorus Pesticides by Their Retention Indices in Reversed-Phase High-Performance Liquid Chromatography

Among the various properties of organic compounds, the distribution coefficients in the 1-octanol/water system (log P) occupy a special place. This parameter, proposed as a measure of the hydrophobicity of organic compounds, is used for various purposes. One of them is predicting the behavior of ecotoxicants in environmental objects. Consideration of the known data on the degradation of pesticides in plants and soil indicates a clearly pronounced dependence of the duration of their detection in such objects on the parameters of hydrophobicity. So, for example, the comparative characteristics of pyrethroids and organophosphorus pesticides (log P values of pyrethroids are on average 2-4 units higher than for FOP) indicate a longer preservation of pyrethroids in various crops (1-2 weeks more), despite significantly lower (several times) norms of expenses. Even within the same class of compounds, the dependence of the duration of registration of pesticides in the soil on their hydrophobicity is well traced.

For example, more hydrophobic FOPs (log P 3-4) are detected 5-15 days longer than less hydrophobic ones (log P 1). In addition to assessing and predicting the behavior of pesticides in various environmental objects, log P values can be used as one of the criteria for selecting new promising plant protection products. Thus, it is believed that the insecticidal activity of organophosphorus compounds also correlates with their hydrophobicity, and thus log P values may be useful in the search for new insecticides. When performing sample preparation using SPE on modified silica gels, as noted in a review of the literature, a number of authors attribute the efficiency of pesticide extraction to their hydrophobicity. Therefore, this parameter is of interest not only to characterize ecological behavior or to search for new promising pesticides, but also from an analytical standpoint. The experimental determination of log P in the 1-octanol/water system is associated with significant difficulties, the main of which should be considered the formation of slowly separating emulsions of both solvents in each other. This leads to an unnecessarily long equilibrium period, the absence of which is manifested in the low interlaboratory reproducibility of log P values for many substances (for some estimates on the example of pesticides, see ). Known methods for determining log P can be divided into two groups - direct and indirect.

Direct methods are based on the direct measurement of the equilibrium concentrations of substances in both (or in one, most often in water) coexisting phases. A classic example of such methods is the widely used "shake flask" method, which allows the determination of log P values in the range from -2.5 to +4.5. However, in a number of cases, the interlaboratory reproducibility of the data obtained with its help reaches ± 1.3 log Р units. Other methods for determining log P are either lengthy or require the use of special equipment. The difficulties of directly measuring log P values have led to the emergence of a large number of indirect methods for estimating them. Some of them are based on the calculation of log Р according to additive schemes (based on the log Р increments of molecular fragments, including using modern software (ACD or CS ChemDraw), others involve the use of two-parameter linear regression equations of the form (8), the coefficients of which are calculated least squares method on datasets for previously characterized substances:

The parameters A include both molecular characteristics - polarizability (molecular refraction), ionization potential, dipole moment, and some physicochemical constants - boiling point, solubility in water (only within the homologous series), as well as experimentally determined retention parameters in reverse-phase HPLC (usually use logarithms of retention factors or capacitance factors log k1). Despite the large number of examples of the characteristics of the hydrophobicity of sorbates in terms of log k (HPLC), such chromatographic invariants as retention indices, which are less dependent on separation conditions than capacity factors, for these purposes are still

Comparison of external standard and standard additive methods for assessing the content of pesticides in plant objects

Assessment of the level of pesticides in plant objects is a crucial and final step in determining trace amounts of ecotoxicants. The literature review noted that two methods of quantitative chromatographic analysis are used for this purpose: the most popular is the external standard method (a variation of the absolute calibration method) and the internal standard method. The widespread use of the external standard method is probably due to the simple determination procedure.

It consists in the analysis of solutions of the standard and the sample obtained from the target sample with further determination of the concentration of the pesticide according to the proportion: where Cx, Cst. - analyte concentrations in test and standard solutions; Mh, Met. - the amount of analyte in the test and standard solutions (if their volumes are equal); Рх, Рst# - the area (height) of the analyte peak in the test and standard solutions. The evaluation of the random component of the error in the results of quantitative determination by the method of an external standard is carried out according to the ratios: where 5СХ, 5Сst. 5MX, 8MST. errors in determining and specifying the amounts of the pesticide in the analyzed and standard solutions (if their volumes are equal); 8PX, SPSCT. - errors in determining the areas (heights) of pesticide peaks in the test and standard solutions. However, at different stages of preparation of samples for chromatographic analysis, significant losses of pesticides can be observed, which leads to a decrease in their concentration in the final test solution, and, as a result, to underestimated results of determinations. The review of the literature also noted that the internal standard method makes it possible to reduce the effect of systematic error on the final results of the analyses. Its advantage in this case would be indisputable if there were no difficulties in choosing internal standards. At the same time, such a variation of the internal standard method as the standard addition method has not yet found its application for assessing the content of pesticides in plant (and other) objects. This method involves the use of the analyte itself as an internal standard. To establish its content in the sample (Cx), it is necessary to analyze two samples: the initial sample and the sample after introducing a known amount of the standard additive into it.

According to a simple proportion (if the analyzed volumes are equal), linking the increase in the chromatographic signal with the addition of the test compound, its initial content in the sample is determined: the determined amount of the analyte in the original sample; MDob. - addition of a comparison sample; Rx, Rx + DOB. are the areas (heights) of the analyte peaks in the samples corresponding to the initial sample and the sample with the additive; m is the mass of the initial sample, V is the volume of the analyzed sample. The random error of the results of quantitative determinations (SMX) by the standard addition method (at 8MDAb « SP and SV« 8 MDAb.) can be estimated by the relation: . Comparison of expressions (15) and (16) shows that the random component of the determination error by the standard addition method at Px Px+add will be greater than the external standard method, since (Px+DDb / (Px+add - Px)» 1, but at Рх+add » Рх and, consequently, Рх+add / (Рх+Add - Рх) « 1 they are comparable in value. In addition, its additional source is a twofold increase in the number of experimental operations during sample preparation. However, a decrease in the effect of systematic error when using the method of standard addition (as well as in the method of internal standard), as a rule, it allows to significantly reduce the total error of determinations.

Kochmola, Nikolai Maksimovich

The search for optimal methods for the analysis of pesticides is one of the most important problems in analytical chemistry. From the modern standpoint, these primarily include capillary gas chromatography (GC), high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and capillary electrophoresis (CE). These methods have a high separating power, which is necessary for the analysis of multicomponent samples, and high sensitivity, which makes it possible to determine pesticides at concentrations of 1 μg/dm 3 and below.

The choice of a specific analysis method is largely determined by the analytical task itself. Typical tasks include the following:

- determination of pesticides at different stages of their production, preparation of finished forms, during their storage;

− determination of residual amounts of pesticides in agricultural products, in soil and in natural waters;

− determination of pesticides in biological samples;

− determination of pesticides in food products, in the atmosphere, in drinking water.

The last two tasks are the most difficult, since they require the simultaneous determination of not known substances, but a set of compounds from the entire list of pesticides used in practice, the number of which exceeds 1000 names. Tasks of this type are sometimes called screening tasks. They are solved mainly using the GC method with mass spectrometric detection (GC-MS), when the identification of pesticides is carried out according to a pre-created library of mass spectra.

Given the wide variety of pesticides, when choosing methods for their determination, preference should obviously be given to "universal" methods. A laboratory operating on the principle of “each substance has its own method of analysis” can achieve high productivity only with respect to a relatively small number of substances. The transition from one group of pesticides to another requires a lot of time to rebuild and calibrate instruments, prepare standards, etc.

Considering chemical-analytical methods from the point of view of their "universality" in relation to the analysis of pesticides, the following remarks can be made.

The TLC method is quite sensitive and easy to perform, however, due to its relatively low resolution, it cannot be “universal”.

The GC method has a very high resolution, but its use is limited by the thermal lability of a number of pesticides and the need to involve various methods of chemical derivatization of many pesticides to increase their volatility.

The capillary electrophoresis method, having a high resolution, does not provide an acceptable concentration sensitivity and requires a very high degree of sample concentration, which is often impossible due to the limited solubility of pesticides.

The HPLC method provides sufficient resolution for solving many problems, does not, as a rule, require preliminary derivatization, and is suitable for the analysis of heat-labile pesticides. In combination with GC, it allows solving almost all problems, and it is these two methods that are most widely used in modern environmental analytical chemistry.

Pesticides, as already mentioned, are classified as priority ecotoxicants, and therefore, must be under constant control in environmental objects. Monitoring of pesticides involves their quantitative determination in a wide range of concentrations, including the background level. Among the methods of analysis that are applicable to the determination of pesticides, first of all, there are high-performance versions of gas and liquid chromatography.

High performance liquid chromatography (HPLC) is one of the most informative analytical methods. It is widely used in all developed countries, but, in comparison with other physicochemical methods of analysis, it requires highly qualified personnel, and the cost of one analysis reaches several tens and even hundreds of US dollars. Thus, simplifying the procedure of HPLC analysis and reducing its cost is an important task.

These shortcomings of HPLC are due to the fact that for each pesticide (or group of pesticides) regulatory documents regulate their own "unique" version of HPLC analysis. This leads to the need to frequently rebuild the chromatograph, which takes a lot of time and requires some experience. In addition, an analytical laboratory that performs analyzes involving many different methods has to maintain a warehouse of expensive columns, organic solvents, and pesticide reference standards.

Pesticides determined in world practice by HPLC include non-volatile and thermolabile compounds. These include atrazine, simazine, chlorprofam, linuron, chlortoluron, alachlor, trifluoalin.

The analysis of pesticides uses special sample preparation methods, which it is useful to consider in more detail.

Liquid-liquid extraction (LLE) is a classic method for extracting pesticides from water samples. Usually, extraction is repeated several times from 500-1000 ml of an aqueous sample in a separating funnel. The most popular solvent is dichloromethane. It is able to extract compounds with different polarity and is easily evaporated. US Environmental Protection Agency (EPA) Methods 8120 and 8140 use LLE with dichloromethane to determine 15 organochlorine and 21 organophosphorus pesticides in water. To extract herbicides - derivatives of carboxylic acids - the source water is acidified to pH<2 и затем экстрагируют неионизованные молекулы диэтиловым эфиром или дихлорметаном.

Classical LLE is difficult to automate, requires large volumes of toxic solvents, and is very time consuming. The separation of solvent layers in the analysis of heavily polluted waters is often hindered by the formation of stable emulsions. In such cases, a single long-term LLE is recommended in a separating funnel with a volume of 1 liter with a solvent heavier than water.

Although the classic LJE has many shortcomings, it continues to improve. This is how microLLE was born, developed as an alternative method for the determination of the herbicide alachlor and its two metabolites. The principle of microLLE - extraction from a large volume of water (400 ml) with a very small volume of solvent (500 µl of toluene) - can be used as a sample preparation for GC analysis without an evaporation step, which is important for the determination of highly volatile compounds. Compared to solid phase extraction, this sample preparation method is faster and cheaper.

A large number of different herbicides (phenylureas, triazines, dinitroanilines, chloroacetamides and uracils) are extracted from food products by mechanical shaking or homogenization with organic solvents such as methanol, acetonitrile, often dichloromethane or ethyl acetate mixed with water, sometimes at an acidic pH.

Highly polar herbicides such as glyphosate are insoluble in most organic solvents and are extracted with water or water with chloroform, sometimes at an acidic pH. In this procedure, other water-soluble components (amino acids, amino sugars, etc.) are also extracted. Their presence interferes with the determination of glyphosates and makes it necessary to purify the extracts, which is most often carried out on ion-exchange chromatographic columns.

Bipyridine pesticides (diquat and paraquat are quaternary ammonium compounds) are usually extracted from matrices by reflux or heating with sulfuric or hydrochloric acid, followed by solid phase extraction and chromatography.

Solid phase extraction (SPE) as a method of sample preparation has been known for 50 years. Its advantages: saving time and solvents, avoiding the risk of emulsion formation, the possibility of isolating trace amounts of the analyte, the possibility of automation. SPE is especially often used in the analysis of natural waters.

SPE is actively used to determine triazine pesticides and their degradation products, hydroxy- s-triazines, herbicides - urea derivatives, N-methylcarbamates and their polar metabolites, organochlorine and organophosphorus insecticides, polar pyrethroid pesticides, triazole and pyrimidine pesticides. SPE methods have been developed for multicomponent mixtures that include a large number of pesticides of various classes. To increase the efficiency of extraction of polar pesticides, columns with a mixture of two sorbents, for example, C18 and Phenyl phases, are sometimes used.

In the SPE of acids on C18 phases, to reduce losses, it is advisable to acidify the sample solution to pH<2. Для ТФЭ неионных соединений иногда применяют графитированные сорбенты и фазы, представляющие собой макросетчатые стирол-дивинилбензольные полимеры. Для пестицидов триазиновой группы, производных мочевины и группы феноксикислот успешно используют картриджи с активированной графитированной сажей Carbopack B, ion exchange resins in acetate form, and a propyl-NH 2 phase. For SPE of organophosphorus pesticides, membrane disks of polystyrene-divinylbenzene type " XAD ».

Supercritical fluid extraction (SCLE) is a relatively new method used to extract substances using special extractants - "supercritical" fluids. Such extractants can be liquid CO 2 , NH 3 , propane, butane, etc. The listed gases pass into a liquid state at high pressures, therefore, SCAE is carried out in autoclaves. After the extraction is completed, the pressure in the autoclaves is reduced to atmospheric pressure, the extractant gas escapes, and only the extracted substances remain in the autoclave. They are dissolved in suitable solvents and the solutions analyzed.

SQLE is used primarily for the analysis of various classes of pesticides in soils, animal and plant tissues. The extraction efficiency is regulated by adding other solvents to the extractant. The most common co-solvent added to carbon dioxide is methanol. Its addition makes it possible to overcome matrix effects, when pesticides, strongly bound to the matrix, are not extracted with pure carbon dioxide. In addition, the addition of methanol or acetone increases the solubility of polar compounds in carbon dioxide.

Direct SLE is rarely used to extract analytes from an aqueous matrix. The limitation of the method is related to the problem of ice formation and the problem of water removal.

At the end of sample preparation, quantitative determination of pesticides is carried out by HPLC and often with a UV detector.

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