Certificate No. 30-08 dated 03/04/2008
FR.1.31.2008.01033

1. Objects of study

This measurement procedure applies to smoked meat, smoked fish and fatty products and establishes the determination of the mass concentration of benzo(a)pyrene by high performance liquid chromatography with fluorimetric detection.

2. Measuring range

MPC for benz(a)pyrene in fatty, smoked meat, smoked fish products is 1 µg/kg.

The method provides obtaining the results of measurements of the mass concentration of benzo(a)pyrene in the ranges presented in Table 1.

Table 1. Measurement ranges of mass concentration of benzo(a)pyrene

Product type	mass range concentration, mcg/kg	MPC, mcg/kg
fat products	0,5 – 2,0	1,0
smoked meat products	0,5 – 2,0	1,0
smoked fish products	0,5 – 2,0	1,0

3. Sample preparation

Sampling, conservation and storage of product samples is carried out in accordance with GOST 7631, GOST 9792, TU and other regulatory documentation governing sampling for specific types of products.

Sample preparation consists of the stages of sampling (the sample is pre-cooled at a temperature of minus 12-18 °C for 30 min), grinding, homogenization with anhydrous sodium sulfate, extraction of the homogenized sample with hexane in a flask using an ultrasonic bath, spontaneous precipitation of a solid precipitate (in for 1-2 min), degreasing the extract in a freezer (silica gel is added to the cylinder, the extract is added, the contents of the cylinder are placed in the freezer), cleaning the supernatant hexane layer by non-retaining solid-phase extraction (on Strata Silica Si-1 cartridges) *, stripping eluate in a stream of air or an inert gas.

* Note. When the concentration of fats in the original analyzed product is less than 5%, a small amount of eluting reagent - ethyl acetate (0.1 ml to 6 ml of the purified extract) must be added to the initial sample extract after freezing.

4. Carrying out chromatographic analysis

4.1. Equipment and conditions for HPLC analysis of benz(a)pyrene calibration solutions, prepared product samples.

For the chromatographic analysis of benzo(a)pyrene, it is necessary to use an isocratic high-performance liquid chromatographic system with fluorimetric detection.

To carry out the analysis, preliminarily prepare calibration solutions from the GRM of a solution of benzo(a)pyrene in hexane or from the GRM of a solution of benzo(a)pyrene in acetonitrile (the solvent is blown off, the standard sample is redissolved in hexane); carry out sample preparation; prepare the device for operation.

Equipment:

liquid chromatograph "Stayer" with a fluorimetric detector;

a personal computer with the installed software "MultiChrome for Windows XP" version 1.5 or 2x.

isocratic mode;

column: Luna C18(2) 150x3.0 mm 3 µm (Phenomenex, USA);

guard column: C18 4x3.0 mm (Phenomenex, USA);

mobile phase: acetonitrile/water solution (75:25);

flow rate: 0.3 ml/min;

loop volume: 10 μl;

temperature: 50°C;

RFU range: 0.01;

detection: fluorimetric (λex: 365±2 nm; λem: 400-460 nm).

Calibration is carried out using calibration solutions (over the entire range of determined concentrations) at least once every two weeks, as well as when using a new batch of reagents, replacing columns, and after repairing the chromatograph.

4.2. Determination of the quantitative content of benzo(a)pyrene in a product sample.

To determine the quantitative content of the product sample component (benzo(a)pyrene), a chromatographic analysis of one of the calibration solutions is carried out, and then a chromatographic analysis of the prepared sample is carried out. For the reliability of measurements, the chromatographic analysis of both the calibration solution and the prepared sample is carried out at least 2 times in a row.

Using the installed software - "MultiChrome for Windows XP" in the report or above the peak (depending on the settings of the "VIEW" options), at the end of the measurement, the result is automatically determined in the form of a concentration in the sample introduced into the chromatograph (but not in the initial sample taken for research!).

To obtain a result, it is necessary to carry out at least two parallel measurements (obtain two chromatograms). The measurement result is taken as the arithmetic mean of the content of benzo(a)pyrene in the concentrate of the analyzed sample C xp, μg/l (calculated from two values ​​of the mass concentration of benzo(a)pyrene in the concentrate of the analyzed sample C 1 and C 2).
Mass fraction of benzo(a)pyrene in the analyzed sample (in the original sample) X, mcg/kg is calculated by the formula:

where:
C xp - the average value of the concentration of benzo(a)pyrene, obtained as a result of chromatography in two parallel measurements [ng/ml];
V 1 – the volume of the initial extract, which is actually equal to the volume of hexane taken for the primary extraction (50 ml);
V 2 - the volume of the extract (part of the original) taken for freezing (30 ml);
V 3 is the volume of the extract (part of the extract after freezing) taken for SPE (6 ml) [ml];
V 5 is the volume of the final extract, part of which is introduced into the chromatograph (about 3 ml) [ml];
K sampling – sampling coefficient, which takes into account the proportion of the mass of the sample of the product (mixed with sodium sulfate) taken for extraction, of the total mass of the sample taken for analysis. In all cases it is equal to 0.736;
K freeze is the loss coefficient of benzo(a)pyrene during freezing. For all categories of the studied products, this coefficient is the same and equals 0.95;
K extra 1 is the coefficient of primary liquid extraction with hexane. For all categories of products under study, it is the same and equal to 0.95;
K TFEextr.2– coefficient of solid-phase extraction of benzo(a)pyrene, equal to 0.95;
m pr. – soil or soil taken for analysis [g].

4.1.2. Method for measuring the mass fraction of benzo(a)pyrene in food raw materials, food products and soil by high performance liquid chromatography

Purpose and scope

The method is intended for the quantitative determination of benzo(a)pyrene (BP) in food raw materials, food products and soil at its mass fraction indicated in Table. 1. The lower limit of the measurement range corresponds to 1/2 of the permissible level (content) of the toxin in products and raw materials, the upper limit - to five times the permissible level.

Benz (a) pyrene is a highly toxic carcinogenic compound, and its permissible content in food products and food raw materials is established by the Sanitary Rules and SanPiN 2.3.2.560-96.

The technique can be applied by institutions of the State Sanitary and Epidemiological Supervision of the Russian Federation, laboratories of other organizations and enterprises related to research, chemical analysis and certification of food products. The methodology is not arbitrage.

Rice. 11.14.

• 1. Naphthalene 9. Chryzen (0.17*)
• 2. Acenaphthene (1.40*) 10. Benz(a)pyrene
• 3. Fluorene (2.60*) 11. Benz(b)fluoranthene (0.26*)
• 4. Phenantrene *2.40*) 12. Benz(c)fluoranthene (0.10*)
• 5. Anthracene (0.13*) 13. Benz(a)pyrene (0.2*)
• 6. Fluoranthene (0.74*) 14. Dibenz(a,b)anthracene
• 7. Pyrene (0.67*) 15. BeH3(g, h, i)nepHaen (0.21*)
• 8. Benz(a)anthracene (0.07*) 16. Indeno(1,2,3-cc1)pyrene (0.26*)

Analysis conditions:

Column: Supelcosil® LC RAS ​​(250 mm x 2.1 mm; 5 µm);

Gradient: Acetonitrile (A) 50-100%, water (B) 50-0%; 200 µl/min Temperature: 25°C Sample volume: 10 µl Detection: Sflu, as per program

Characteristics of measurement error

The limits of the relative error (±5) of the measurement result of the mass fraction of BP (with a confidence level of 0.95) are indicated in Table. one.

Table 1. Analyzed objects, ranges and characteristics of measurements

Product group (analyzed object)	Allowed content ( IV X), mg/kg	Measurement range of BP mass fraction, mg/kg	Coefficient extraction,	Relative error limits (±8), %
Smoked meat, fish and fat products

Measuring instruments, auxiliary devices, materials and reagents

Measuring instruments

Liquid chromatographs:

• - Microcolumn "Milichrome-5", TU 25-7405.0009-89, version 3 with a fluorimetric (FlD) detector (Option 1).
• - Isocratic or gradient liquid chromatograph, for example, "Kpayeg" (Germany), State Register of Measuring Instruments of the Russian Federation 16848-97 or Chromatographic Attachment "VEZHKh-3", LLP "Lumex" (St. Petersburg), equipped with FLD "Fluorat-02 -2M", LLP "Lumex", State Register of Measuring Instruments of the Russian Federation 14093-99 (Option 2).
• - Isocratic or gradient liquid chromatograph with PLD of any type, for example "Kpaerg" (Germany), State Register of Measuring Instruments of the Russian Federation 16848-97 (Option 3).

Chromatographic columns "Diasfer-110-S 16", TU 4215-001-05451931-94, CJSC "BioKhimMak ST" (Moscow), with standard sizes corresponding to the variant of the chromatographic system:

• - 2 x 80 mm, dp = 5 - 7 µm (Option 1)
• - 2 x 150 mm, dp = 5 - 7 µm (Option 2)
• - 4 x 150 mm, dp = 5 - 7 µm (Option 3)

Hardware and software complex "MultiChrom-Spectrum", TU AZHRC

3.036.001, CJSC Ampersend (Moscow), or any other software that allows calibration and quantitative determination by the external standard method.

GSO 7515-98 of the composition of a solution of benzo(a)pyrene in acetonitrile with a mass concentration of benzo(a)pyrene 100 μg/cm 3, AOZT "Ekros" (St. Petersburg).

GSO 7064-93 composition of a solution of benzo(a)pyrene in hexane with a mass concentration of benzo(a)pyrene 100 µg/cm 3 , AOZT "Ekros" (St. Petersburg).

Scales laboratory electronic 4 cells. accuracy model VLE 134, GOST 24104-88 or others.

100 µl microsyringes from Hamilton, model Microliter #1710 or equivalent.

Micropipettes 0.5; 0.2; 0.1 µl; GOST 20292-74.

Dimensional cylinders 2-25.2-50, 2-100 and 2-500, GOST 1770-74.

Volumetric flasks 2-10-2, 2-100-2, GOST 1770-74.

Graduated pipettes 1, 2, 5, 10 cm 3 , GOST 29227-91.

Reagents and materials

Acetonitrile for liquid chromatography, OP-3 high purity, TU 6-09-14-2167-84, rectified.

Bi-distilled water, TU 6-09-2502-77.

Hexane, chemically pure, TU 6-09-3375-78, dried over Na 2 S 04 , rectified.

Benzene, chemically pure, GOST 5955-75, dried over Na 2 S 04 , rectified.

Anhydrous sodium sulfate, chemically pure, GOST 4166-76.

Concentrating cartridges "Diapak": A-3, P-3, C; TU 4215-002-05451931-94, ZAO BioKhimMak ST (Moscow).

Auxiliary devices

System for filtration and degassing of eluents CJSC "BioKhimMak ST" (Moscow) or others.

1.8 and 5.0 cm 3 glass vials for calibration and analysis solutions with screw caps and Teflon gaskets from Supelco, catalog numbers 2-6951, 2-7037 and

2-7039, or equivalent.

Micromixer PPE-3, "Ekros" (St. Petersburg).

Rotary evaporator IR-1M2, TU 25-1173.102-84 or others.

Sharp-bottom flasks with stoppers with a capacity of 25, 10 and 5 cm 3, GOST 25336.

A device for blowing off solutions in a stream of nitrogen, equipped with a thermostatically controlled aluminum block (air-dry bath) SP "BioMark" (Lvov) or others.

Membrane filters with dp = 0.4-0.5 µm.

A device for creating a vacuum of about 7 mm Hg. Art. (water jet pump, GOST 25336; Begemot water ring vacuum pump, UVK-RK2/1, CJSC BioKhimMak ST, Moscow).

Vacuum sample preparation device (vacuum manifold) or other with sample receivers with a capacity of at least 10 cm 3 .

Buechner flask, Bunsen funnel with a capacity of at least 500 and 200 cm 3, respectively, GOST 1770.

Separating funnel with a capacity of 100 and 500 cm 3, GOST 25336.

Flat-bottomed conical flasks with stoppers with a capacity of 50, 100 and 250 cm 3, GOST 25336.

Pear-shaped flasks with stoppers with a capacity of 50 and 100 cm 3, GOST 25336.

Conical funnel with a diameter of at least 10 cm, GOST 1770.

Filter paper of the "blue tape" type.

Cotton wool medical non-sterile, cotton.

Measurement method

The technique includes the following main procedures:

• - primary extraction with hexane and re-extraction into acetonitrile BP from a sample of a smoked product;
• - primary extraction of BP with a mixture of acetonitrile-water from a sample of grain or soil;
• - concentration and purification of the primary extract by solid-phase extraction;
• - dilution of the prepared sample extract with a mixture of acetonitrile-water;
• - calibration of the chromatograph according to solutions with a known value of the mass concentration of BP;
• - analysis of the prepared sample extract solution by high performance liquid chromatography (HPLC) with registration of the fluorescence signal;
• - identification of the determined BP by the retention parameters;
• - calculation of the mass concentration of BP based on the registered analytical signal and calibration characteristics;
• - calculation of the mass fraction of BP, based on the mass concentration of BP, the mass of the food sample and the volume of the solution of the prepared extract.

Safety requirements

When working with used chemical compounds, it is necessary to comply with the safety requirements established for work with toxic, caustic and flammable substances, GOST 12.1.018-86 and GOST 12.1.004-76, fire safety requirements, GOST 12.1.004-76.

If BP solutions get on the skin" or surfaces of objects, they must be treated with water and detergent, and then with ethyl alcohol. Solutions should be stored in a refrigerator in sealed packaging.

When operating the system for HPLC and carrying out the corresponding measurements, it is necessary to observe the rules of electrical safety, GOST

12.1.019-79 and operating instructions for the device.

Operator Qualification Requirements

Persons allowed to work:

• - qualified as a chemical engineer or chemical technician;
• - having experience in a chemical laboratory;
• - who have completed relevant training courses and internships in laboratories accredited to perform analyzes using HPLC;
• - received positive results during the performance of control operations.

Measurement conditions

Sample preparation, preparation of solutions, preparation and performance of measurements are carried out at an ambient temperature of 18-25 ° C, atmospheric pressure of 84.0-100.7 kPa (630-800 mm Hg), air humidity of not more than 80% (at temperature 25 °C).

When measuring in the laboratory, the following conditions must be met: mains voltage 220 ± 10 V, mains frequency 50zh 1 Hz. Measurements are carried out under conditions recommended by the description and operating instructions of the device.

Preparing to take measurements

Glassware

Before further use, used glassware is rinsed with the last of the solvents used and thoroughly washed with hot water with any washing powder, rinsed successively with distilled and bidistilled water and dried. Clean dishes are stored by closing with a cork or cotton swab.

Sampling, storage and handling

Sampling and averaging is carried out in accordance with the regulatory documents for each type of product (GOST 13586.3-83, GOST 27668-88, GOST 9792-73, GOST 7631-85). The determined BP is extracted from samples of smoked products by extraction with dry hexane, after evaporation of which it is re-extracted into acetonitrile. When extracting BP from grain or soil samples, a mixture of water-acetonitrile (16:84) is used. The subsequent concentration and purification of the primary sample extract containing BP, regardless of the nature of the initial product, is carried out in accordance with the complex scheme of solid-phase extraction using three concentrating cartridges Diapak A-3, P-3, S.

Prepared samples (extracts of samples) are dissolved in a mixture of acetonitrile-water (70:30).

Each measurement of the mass fraction of BP includes the preparation and chromatographic analysis of at least two samples.

Preparation of solvent mixtures

Solvent mixtures are prepared by the volumetric method in graduated cylinders. The required volumes of acetonitrile and water are measured with separate graduated cylinders and then mixed. Extractant A: acetonitrile-water (84:16).

Preparation of extractants

To prepare mutually saturated acetonitrile and hexane, shake about 300 cm 3 of acetonitrile and 100 cm 3 of hexane in a separating funnel with a capacity of 500 cm 3 . After separation of the solvents, the layers are taken separately: the lower layer (acetonitrile saturated with hexane - extractant B) and the upper layer (hexane saturated with acetonitrile - extractant C), the interphase is discarded.

Preparation of eluents

For HPLC measurements, acetonitrile-water mixtures are prepared in the following ratios: (90:10) - eluent 90, (84:16) - eluent 84, (80:20) - eluent 80, (70:30) - eluent 70. Ready eluents are filtered through a membrane filter and vacuum or thermal degassing is carried out.

Preparation of calibration solutions

GSO of the composition of the BP solution in acetonitrile (see above) is diluted with a mixture of acetonitrile-water (7:3) before use. Pipette taken

1.0 cm 3 of the stock solution is placed in a volumetric flask with a capacity of 100 cm 3 and the solvent is added to the mark. Next, certain volumes of the resulting solution are taken with a pipette, placed in a volumetric flask with a capacity of 10 cm 3 and the solvent is added to the mark. The corresponding volumes of solutions used for dilution and concentrations of calibration solutions 2-5 (Option 1) and 3-7 (Options 2, 3) are shown in Table. 2. When using GSO of the composition of the BP solution in hexane (see below), after evaporating the solvent under the conditions given below, the dry residue is redissolved in acetonitrile and diluted as described above, taking into account the value of the certified concentration of GSO.

Table 2. Solutions for calibration in the analysis of benzopyrene (BP)

Mass concentration of BP (certified value of GSO), mcg/cm 3	Initial dilution solution		calibration
				Mass concentration of BP, µg/cm3

*Not used for direct calibration of the chromatographic system.

Preparation of the chromatographic system

The chromatograph is turned on and prepared for operation in accordance with its description and operating manual. Install the column "Diasfer-110-C 16" with standard sizes in accordance with the chromatograph option (see above). The eluent with the highest concentration of acetonitrile is pumped through the chromatographic system until the baseline of the detector is stabilized, and then it is conditioned under the initial conditions of the gradient according to the relevant sections of "Analysis Conditions".

Preparation of concentrating cartridges

Concentrating cartridges Diapak A-3 and P-3 are prepared for work as follows:

• 1. 3 cm3 of dry Diapak A or P sorbent is poured into a 10 cm 3 polypropylene housing with a replaceable filter in the lower part. The upper empty part of the housing is used as a funnel for applying a sample or eluent.
• 2. Clamp the cartridge vertically in a suitable vacuum device and tap to form a flat, horizontal top layer of sorbent. For the final preparation of the Diapak A-3 cartridge, it is enough to fix the sorbent layer with a small cotton swab.
• 3. To prepare the Diapak P-3 cartridge, the sorbent is washed successively with 10 cm 3 of benzene, acetone and extractant A at low vacuum (drip rate no more than 1-2 drops per second), preventing air from entering the sorbent. After filling the cartridge with acetone, the sorbent is allowed to settle, the upper polymer filter is introduced, it is compacted over the upper layer of the sorbent, and washing is continued. Upon reaching extractant A level 2-3 cm above the filter, washing is stopped and the cartridge is sealed with a bottom plug and a top cover (for storage). In case of accidental drying, the cartridge is washed extractant A. Before applying the sample, the plugs are removed and, with a weak vacuum, the remains of the water-acetonitrile mixture are passed to the level of the upper filter, then the test solution is immediately poured. Regeneration of the reusable cartridge Diapak P-3 is carried out according to a similar scheme, excluding the removal of the upper filter.

The concentrating cartridge Diapak C is prepared for work as follows:

• 1. Ready-made polypropylene capsule with 1 cm3 of Diapak S sorbent is sealed with plugs. After removing the plugs, the cartridge is prepared for work by passing 5 cm 3 of hexane through it with a syringe at a drip rate of 1-2 drops per second.
• 2. The sample is applied by gravity, using an empty polypropylene body with a volume of 10 cm 3 as a funnel, tightly fixed in the upper fitting of the Diapak C capsule.

Sample preparation for measurements

Hexane extraction of benzo(a)pyrene from a sample of a smoked product A sample of 10.0 g of a sample of a smoked product is ground in a mortar with 30 g of anhydrous sodium sulfate. The mixture is transferred quantitatively to a 100 cm 3 flat-bottomed flask and extracted with 40 cm 3 of hexane for at least 30 minutes with stirring. The primary hexane extract of the total fat of the sample is decanted and passed through 10 g of anhydrous sodium sulfate into a stripping flask. Repeat the extraction procedure twice with two volumes of 20 cm 3 of hexane and pass portions of the extract through a desiccant into the same distilling flask. Evaporate hexane on a rotary evaporator at a temperature not exceeding 35 ° C until the smell disappears.

The extract of total fat is dissolved in 20 cm 3 extractant B, transfer quantitatively into a graduated cylinder with a capacity of 50 cm 3 and bring the volume of the solution to 40.0 cm 3 with the same solvent.

20.0 cm 3 of the resulting solution is transferred into a separating funnel with a capacity of 100 cm 3 and the BP is re-extracted into acetonitrile with three volumes of 20 cm 3 extractant B. Each time, achieving the most complete separation of the phases, the lower layer is taken (acetonitrile extract of BP) and evaporated on a rotary evaporator at a temperature not exceeding 50 °C up to a volume of 10-15 cm 3. Quantitatively (using acetonitrile) transfer the solution into a measuring cylinder with a capacity of 25 cm 3, bring the volume to 21.0 cm 3 with acetonitrile, add 4.0 cm 3 of bidistilled water and mix thoroughly the resulting water-acetonitrile extract of BP.

Extraction of benzo(a)pyrene from a sample of grain or soil with a mixture of acetonitrile-water

A portion of 10-25 g of the sample is transferred into a flat-bottomed flask, 50-125 cm 3 are added extractant A, strictly observing the ratio of 1:5 sample of the product to the volume of the extractant, and stirred for 1 hour. filtered water-acetonitrile extract of BP through a paper filter on a Buchner funnel under vacuum and squeeze out the precipitate on the filter.

Pre-purification and concentration of the primary extract of the grain or smoked product

Pass through the cartridge Diapak A-3 25.0 cm 3 and then 3 cm 3 extractant A at a drip rate of 2-3 drops per second into a receiving flask.

The collected eluate is passed through the prepared cartridge Diapak P-3, and then 5 cm 3 of acetonitrile at a drip rate of 1-2 drops per second, discarding the washouts. The target fraction containing BP is eluted from the cartridge with a mixture of benzene-acetonitrile (1:1) in a volume of 7 cm 3 at a dripping rate of 1-2 drops per second into a distilling flask. The eluate is evaporated on a rotary evaporator at a temperature not exceeding 50 °C, 0.5 cm 3 of hexane is added to the flask and thoroughly shaken on a micromixer until the dry residue is completely dissolved.

Preliminary purification and concentration of the primary soil extract

Pass through the cartridge Diapak A-3 5.0 cm 3 water-acetonitrile extract of BP, then 3 cm 3 extractant A at a drip rate of 2-3 drops per second into a receiving flask. Transfer the eluate to a graduated cylinder with a capacity of 25 cm 3, rinse the flask with two volumes of 5 cm 3 extractant A and bring the volume of the solution in the cylinder to 20 cm 3 .

5.0 cm 3 of the diluted eluate are passed through the prepared cartridge Diapak P-3, and then 5 cm 3 of acetonitrile at a dripping rate of 1-2 drops per second, discarding the washouts. The target fraction containing BP is eluted from the cartridge with a mixture of benzene-acetonitrile (1:1) in a volume of 7 cm 3 at a dripping rate of 1-2 drops per second into a distilling flask. The eluate is evaporated on a rotary evaporator at a temperature not exceeding 50 °C, 0.5 cm 3 of hexane is added to the flask and thoroughly shaken on a micromixer until the dry residue is completely dissolved.

Fine purification of the extract

0.5 cm 3 of the sample solution in hexane is applied to the prepared Diapak C cartridge by gravity, then the flask is washed with two portions of 0.5 cm 3 of hexane each and sequentially applied to the cartridge, discarding all washes. BP is eluted with benzene in a volume of 2.0 cm 3 at a rate of 1-2 drops per second into a stripping flask and evaporated on a rotary evaporator at a temperature not exceeding 50 °C. Dissolve the dry residue of the extract of the BP sample in a mixture of acetonitrile-water (7:3), the volumes of which are indicated in the sections "Measurement conditions" for each variant of the chromatographic system (see below).

Graduation and measurements

Chromatograph calibration

The chromatograph is calibrated by successive input (under the conditions of BP measurement) of the nominal volume of calibration solutions (Table 2) in ascending order of their mass concentrations. Each solution is injected into the chromatograph at least twice. With the correct setting of the chromatographic system, the height of the peak on the chromatogram of the calibration solution with the lowest concentration should exceed the baseline noise level by at least 10 times.

After mathematical processing of the chromatograms, the retention parameters and peak areas are fixed and calibration characteristics (GC) are built, reflecting the dependence of the average value of the peak area on the mass concentration of BP in the calibration solution.

Control the correctness of the construction of the calibration characteristics.

The calibration characteristic is rebuilt when changing columns, after carrying out maintenance and preventive maintenance, with negative results of monitoring the stability of GC (see below).

Determination of benzo(a)pyrene

Measurement conditions (Option 1)

For analysis, the prepared extract of the BP sample is dissolved in 0.1 cm 3 of an acetonitrile-water mixture.

The modes of operation of FMD and UVPA are set from the computer keyboard in accordance with the User's Manual (HSS "MultiChrome-Spectrum") and are controlled on monitors in the following form:

Fluorometric detector

• excitation wavelength 296 nm;
• emission wavelength - light filter No. 2 (more than 380 nm);
• measurement time 0.2 s.

Automatic dispenser

• regeneration volume 0.4 cm 3 ;
• sample volume 0.04 cm 3 ;
• flow rate 0.15 cm 3 /min;
• recruitment speed 0.3 cm 3 /min;
• BP retention time 11 min.
• the composition of the eluents in the vessels and the scheme for compiling the acetonitrile gradient are presented in Table. 3.

Table 3 eluents

Measurement conditions (Option 2)

For analysis, the prepared (see above) BP sample extract is dissolved in 0.5 cm 3 of an acetonitrile-water mixture.

• filter on the excitation line - "X4";
• filter on the emission line - "KhZ";
• sample volume 0.02 cm 3 ;
• sensitivity is average;
• smoothing - 4;
• the “background” level is chosen based on the results of registration of a test chromatogram of calibration solution No. 3.

• flow rate 0.2 cm 3 /min;
• eluent 84;
• BP retention time is about 12 min.

Gradient Split Mode:

• flow rate 0.25 cm 3 /min;
• eluent 100 in eluent 70 in 20 minutes;

Measurement conditions (Option 3)

For analysis, the prepared extract of the BP sample is dissolved in 0.5 cm 3 of an acetonitrile-water mixture.

• excitation wavelength 375 nm;
• emission wavelength 405 nm;
• flow rate 0.8 cm 3 /min;
• sample volume 0.02 cm 3 ;
• time constant 1.0 s.

Isocratic separation mode:

• eluent 84;
• BP retention time is about 12 min;

Gradient Split Mode:

• linear gradient from 30 to 70% eluent 100 in eluent 70 in 20 minutes;
• BP retention time is about 14 min.

Acquisition and processing of chromatograms

The sample extract solution is introduced into the chromatograph twice. The identification of BP is carried out on the basis of a comparison of the peak retention parameters in the chromatograms of the sample extract and calibration solutions. Approximate retention parameters are indicated in the sections "Measurement conditions". Reliable identification of the analyzed compound corresponds to the difference between the values ​​of the retention parameters for the calibration solution and the sample, which does not exceed 0.2 min.

Dilution of the extract solution

Carried out in the event that the mass concentration of the determined BP exceeds its highest mass concentration in the calibration solutions. The extract solution is diluted twice (dilution ratio, dil = 2) by taking equal volumes of this solution and a mixture of acetonitrile-water (70:30) and mixing the latter. In the event that a single dilution does not eliminate the "off scale", the procedure is repeated (the degree of dilution, dil = 4).

Processing of measurement results

Calculate the average value of the peak area (chromatograph output signal) for two injections of the sample extract solution into the chromatograph. Control the convergence of the output signals (see below).

According to the calibration dependence, the value of the mass concentration of BP in the solution is found, corresponding to the average value of the peak area.

The mass fraction of BP (I^), mg/kg, in the i-th sample (the result of determination) is calculated by the formula

where Cbp is the mass concentration of the analyte in the solution of the extract of the i-th sample of BP, μg/cm 3 (calculated according to the calibration dependence, based on the average value of the peak area); Vp- the volume of the solution of the extract of the /-th sample of BP, cm 3; R- the degree of extraction of BP at the stage of sample preparation according to Table. 2; M eq- mass of the part of the sample corresponding to the proportion of water-acetonitrile extract of BP used for purification and subsequent chromatographic determination - equivalent mass of the sample, which is 5.0 g (grain, smoked products) or 0.25 g (soil).

In the case of dilution of the extract (see above), the mass fraction of BP ( W,-, mg/kg) in the /-th dimension is calculated by the formula

where wj- the value obtained by the formula (II. 1), dil - the degree of dilution (see above).

Calculate the average value of the mass fraction of BP for two samples (analysis result):

Control the convergence of the results of determining the mass fraction of BP (see below).

Registration of measurement results

The result of the analysis (measurement) of the mass fraction of BP in the object being determined is presented in the form

where W- mass fraction of BP, calculated by formula (II.3).

If the BP is not detected, the measurement result is presented in the form

where P "ds is the permissible content of BP according to Table 1.

MVI error control

Convergence control of chromatograph output signals

The control is carried out during the calibration and analysis of each sample in relation to the output signals (values ​​of the areas of the BP peaks on the chromatograms) obtained with two injections of the solution into the chromatograph. The control result is considered satisfactory if the range of output signals, referred to the arithmetic mean, does not exceed 8%.

Control of the correctness of the construction of the calibration characteristic

Control is carried out at each calibration. The control result is recognized as satisfactory if the conditions for each "th calibration solution are met

where sj- average value of the BP peak area for the y "th calibration solution, c.u.; S)- the value of the peak area corresponding to the calibration characteristic of the mass concentration of BP in the y "th calibration solution, c.u.

Controlling the stability of the calibration characteristic

Control is carried out daily before starting work with the analyzed samples using a control solution, which is used as a calibration solution with a mass concentration of BP corresponding to its permissible content in the analyzed object.

The control result is recognized as satisfactory if the condition is met

where C ki - the value of the mass concentration of BP in the control solution, found by the calibration characteristic for the average value of the peak area, µg/cm 3 ; With to - the value of the mass concentration of BP in the control solution according to the table. 2.

Control of convergence of determination results

The control is carried out at each analysis (measurement). The result of the control is considered satisfactory if the range of the results of the determinations, referred to the arithmetic mean (the result of the analysis), does not exceed 10%.

Error control by additive method

Control is carried out:

• a) before the start of the application of this MVI - without fail;
• b) when doubtful results of determining the mass fraction of BP appear;
• c) in accordance with the plans of intralaboratory control;
• d) at the request of organizations that control the activities of the laboratory.

The additive is formed on the basis of the calibration solution. Additive value (D, mg/kg) is chosen so that the mass fraction of BP in the sample increases by 1.5-2.5 times. The calculation is carried out according to the formula

where C D- mass concentration of BP in the calibration solution, µg/cm 3 ; Vo- the volume of the BP calibration solution introduced into the sample, cm3; M fw- equivalent weight of the sample taken for analysis, g.

The additive is introduced into the primary extract of a grain or soil sample in the form of a calibration solution prepared in accordance with Table. 2. The additive is introduced into the primary extract of the smoked product in the form of a solution of a similar concentration in hexane. To do this, the GRM of the BP composition in hexane (see above) is diluted (taking into account the correction for the certified concentration value) in accordance with the procedure for preparing calibration solutions (see above), using hexane as a solvent and obtaining a solution of the required concentration. When using GSO composition of BP in acetonitrile after evaporation of the solvent, the dry residue is redissolved in hexane and diluted with hexane, as described above.

The analysis of two samples with the natural content of BP and two samples with the addition of BP is carried out under the same conditions (one instrument, one calibration characteristic, one operator). The control result is recognized as satisfactory if the condition is met

where W D- mass fraction of BP in samples with additive, mg/kg; W- mass fraction of BP in samples without additive, mg/kg ( W D and W- average values ​​of the mass fraction for two samples with positive results of the control of convergence).

An illustration of the application of this technique in practical ecoanalytics can serve as a chromatogram and a calibration graph for the determination of benzo(a)pyrene in food products (Figures 11.15 and 11.16).

The results of the control of the error in the determination of benzo(a)pyrene in objects by the method of additions with fluorimetric detection 375 Ex/405 Et (Variant 3).

The level of the additive corresponds to 0.5 of the allowable content of benzapyrene (0.0005 mg/kg - grain and smoked products, 0.01 mg/kg - soil).

Rice. 11.15.

Graduation for component: BaP Correlation coefficient: 0.999667 Response: Area

Reference channel 365 Ex/405 Em

Formula Y = Ki X

Values ​​100 ( W D - W-D)/D are 10 and 16% for samples 1 and 2, respectively. The determined compound in the initial sample of wheat flour is present in the amount of 0.00009 kg/kg.

Sample 1 - 0.00064 mg/kg Sample 2 - 0.00067 mg/kg

• In the general case, the calibration characteristic has the form S = AC + B, where S is the peak area, c.u.; C is the mass concentration of the pollutant, µg/cm3; A and B are coefficients.

Polycyclic aromatic hydrocarbons: physicochemical properties and biological effects. Review of methods for the determination of benzapyrene. Determination of benzapyrene in water by high performance liquid chromatography with fluorescent detection.

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This course work contains 30 pages, 1 section, 4 subsections, 14 paragraphs, 6 tables and 9 figures. At the end of the course work is a list of references, consisting of 14 items.

The object of my research is benzo(a)pyrene, its properties, carcinogenic effect, as well as methods for its determination. The course work presents such methods as gas chromatography with mass spectroscopic detection and high performance liquid chromatography with fluorescent detection. In addition, detectors were considered that are suitable for recording the analytical signal of benzo(a)pyrene and the rationality of using one or another detector was substantiated.

Also in the course work are presented methods for the determination of benzo(a)pyrene in water, consisting of sample preparation, calibration of the chromatograph, analysis and data recording. The paper presents chromatograms obtained by these methods.

Keywords: POLYCYCLIC AROMATIC HYDROCARBONS, BENZO(A)PYRENE, HIGH PERFORMANCE LIQUID CHROMATOGRAPHY, GAS CHROMATOGRAPHY, FLUORESCENT DETECTION.

Tazi course work including 30 pages, 1 section, 4 subsections, 14 points, 6 masi and 9 numbers. At the edge of the cursive work, a list of literature is given, starting from point 14.

Celta on my study of benzo (a) pyrene, negate properties, carcinogenic effects, and somehow method for it is not determined. Course work including such methods as gas chromatography with mass-spectral curve and high-efficiency chromatography with fluorescence curve. This is what detectors are considered to be, which are suitable for analytical reception of a signal from benzo(a)pyrene and justified use on the detector.

Basically, in the course of work on the basin, we present the methods for determining benz (a) pyrene in water, consisting of preparation on a sample, calibrating on a chromatograph, analysis and recording on data. Charter presenting data, obtaining according to the methodology.

Key thoughts: POLYCYCLIC AROMATIC WATER, Benz(A)PYRENE, HIGHLY EFFICIENT TECHNA CHROMATOGRAPHY, GAS CHROMATOGRAPHY, FLUORISCENT OPENING.

The course work was given to cover 30 sides, 1 split, 4 subfolders, 14 points, 6 tables and 9 drawings. In the end of the course work, the list of literature, which consists of 14 points.

About "my research on benz (a) pyrene, its power, carcinogenicity, as well as the method of its identification. In the course work, such methods as gas chromatography with mass spectroscopic detection are highly effective, but chromatography is highly effective and fluorescent. detectors that are suitable for registering an analytical signal to benzo (a) pyrene and the rationality of the other detector is substantiated.

Also, in the course work, the methodology for the designation of benzo(a)pyrene in water is presented, which consists of sample preparation, chromatograph calibration, analysis and registration of data. The robots are represented by chromatograms, taken from these methods.

Key words: POLICYCLIC AROMATIC CARBOHYDRATES, Benz(A)PYRENE, HIGHLY EFFICIENT RIDINNA CHROMATOGRAPHY, GAS CHROMATOGRAPHY, FLUORESCENT DETECTION.

SYMBOLS

INTRODUCTION

1. LITERATURE REVIEW

1.1.1 General information

1.1.2 Origin of PAHs

1.1.4 Biological action

1.1.5 Benz(a)pyrene. General information

1.2 Methods for the determination of benzapyrene

1.2.1 Gas chromatography

1.3 Determination of benzapyrene in water by HPLC

1.3.2 Sample preparation

1.3.3 Graduation

1.3.4 Performing HPLC analysis

1.3.5 Recording and processing of data

1.4.2 Quantification of BP

BIBLIOGRAPHY

SYMBOLS

PAHs - polycyclic aromatic hydrocarbons

BP - benzo(a)pyrene

MPC - maximum permissible concentration

MPC SS - average daily maximum allowable concentration

HPLC - high performance liquid chromatography

GC - gas chromatography

LC - liquid chromatography

NPC - normal phase chromatography

RPCH - reverse phase chromatography

LLE - liquid-liquid extraction

OFS - reversed-phase sorbent

TLC - thin layer chromatography

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) belong to the group of persistent organic pollutants. They have pronounced carcinogenic properties. One of the most dangerous representatives of PAHs is benzo(a)pyrene (BP).

Benz (a) pyrene was discovered in 1933, later, in 1935, studies were carried out confirming its carcinogenicity. Today, benzo(a)pyrene is classified as a carcinogen of the 1st hazard class. It has mutagenic properties. Even a small concentration of BP negatively affects the human body. The concentration of BP in the air exceeding the maximum allowable concentration (MAC) with prolonged exposure can cause lung cancer. Therefore, the problem of its detection and definition is acute. Based on its physical and chemical properties, a number of similar methods for its determination were developed, differing only in the stages of sampling and sample preparation. The purpose of my work was to get acquainted with the properties of PAHs and BP, to study methods for separating PAHs and methods for determining BP.

1. LITERATURE REVIEW

1.1 Polycyclic aromatic hydrocarbons (PAHs)

1.1.1 General information

PAHs are high-molecular organic compounds of the benzene series, numbering more than 200 representatives. They contain from 2 to 7 benzene rings. PAHs are widely distributed in nature and are stable over time. They have carcinogenic and mutagenic activity. Due to their toxicity and carcinogenic properties, they are classified as priority pollutants. The determination of PAHs is used in ecological and geochemical studies. The most toxic of them are 3, 4-benz(a)pyrene and 1, 12-benzperylene, which are especially often determined in environmental objects.

1.1.2 Origin of PAHs

PAHs are an undesirable by-product of burning fossil fuels. They are formed in nature due to abiogenic processes. Thousands of tons of PAHs released from the humic components of the soil enter the biosphere every year. But most of these carcinogens come from man-made processes.

Coal is considered to be a mixture of a huge amount of polycondensed aromatic benzene nuclei with a minimal hydrogen content. When these substances are burned in furnaces, power plants, internal combustion engines, these compounds decompose. At low combustion temperatures and insufficient supply of atmospheric oxygen, reactive acetylene and aliphatic hydrocarbons are formed. Acetylene polymerizes to butadiene, which then forms the nucleus of an aromatic hydrocarbon. When added to existing aromatic nuclei, PAHs are produced.

Incomplete combustion produces particles of carbon - soot. PAHs are adsorbed on its surface and enter the environment.

1.1.3 Physical and chemical properties of PAHs

Most PAHs are crystalline compounds (with the exception of some naphthalene derivatives) with a high melting point. PAHs are poorly soluble in water. When moving to organic solvents, their solubility increases and depends on their molecular weight. As a rule, with an increase in the number of aromatic rings and alkyl radicals, the solubility of PAHs decreases.

Most PAHs intensely absorb UV radiation (300–420 nm) and rapidly photooxidize in the atmosphere to form quinones and carbonyl compounds.

1.1.4 Biological action

PAHs enter the body through the respiratory tract, skin or digestive tract.

The type of interaction of PAHs with the body mainly depends on the hydrocarbon itself. Basically, when PAHs enter the body, enzymes form an epoxy compound that reacts with guanine, which interferes with DNA synthesis, causes disorders, or leads to mutations that contribute to the development of cancer.

One of the most toxic PAHs, as mentioned earlier, is BP. In addition, the carcinogenic activity of PAHs is 70-80% due to the influence of BP. Therefore, the presence of BP in food products can be used to judge the presence of other PAHs.

1.1.5 Benz(a)pyrene. general characteristics

Benz (a) pyrene (C 20 H 12) is a chemical compound, a representative of the family of polycyclic hydrocarbons (Fig. 1.1), a substance of the first hazard class. It is formed during the combustion of hydrocarbon liquid, solid and gaseous fuels (to a lesser extent during the combustion of gaseous fuels).

Figure 1.1 Structural formula of benzo(a)pyrene.

BP is yellow plates or needles. It is highly soluble in non-polar solvents, for example, in toluene, benzene, xylene. It is slightly less soluble in polar solvents, but practically insoluble in water.

BP accumulates mainly in soil, less often in water. From the soil, it enters the plants, continuing its movement along the trophic chain. At each next level, the content of benzo(a)pyrene increases by an order of magnitude.

BP is a typical chemical carcinogen and is dangerous to humans even in minimal concentrations, since it has the property of accumulating in the human body. MPC of benzo(a)pyrene in different objects is presented in Table 1.1. In addition, BP has mutagenic properties, i.e. it can cause mutations.

Table 1.1 MPC of benzo(a)pyrene in various environments

Object name	MAC, mcg/kg
smoked products
Cereals
Drinking water
Water reservoirs

In the air, the average daily maximum allowable concentration (MPC CC) is 0.1 µg/100m 3 .

1.2 Methods for the determination of benzo(a)pyrene

The main methods for determining PAHs are reversed phase high performance liquid chromatography (HPLC) with fluorimetric or spectroscopic detection and gas chromatography (GC) with mass selective, flame ionization, electron capture or photoionization detection.

To determine the BP, the method of luminescent spectroscopy based on the Shpolsky effect is also used. The essence of the effect lies in the fact that at low temperatures some polyatomic molecules give high-resolution quasi-linear luminescence spectra. The advantage of this method is the low demands on the degree of purification and sensitivity. But the complexity of the hardware is a significant limitation to the definition of BP.

Chromatography is a method of separation, analysis and physicochemical studies of substances based on the movement of a zone of a substance along a sorbent layer in a mobile phase flow with multiple repetitions of sorption and desorption acts. Separation occurs due to the difference in the distribution constants of individual substances between the two phases. The most important feature of chromatography is the dynamic nature of the process, in which gradients occur in the concentration distribution of molecules or particles.

The general scheme for separating the components of the mixture is shown in Figure 1.2.

Figure 1.2 Scheme of the implementation of the chromatographic process

The advantages of chromatographic methods include the possibility of simultaneous implementation of a large number of parameters characterizing the separation, identification, and quantification of components in a mixture. Thus, chromatography is a multichannel source of information.

Depending on the state of aggregation of the mobile phase, chromatographic methods are divided into gas and liquid chromatography.

Gas chromatography, in turn, depending on the state of aggregation of the stationary (stationary), includes gas-liquid and gas-solid-phase chromatography.

Liquid chromatography is divided into liquid-liquid, liquid-solid phase and liquid-gel.

1.2.1 Gas chromatography

GC is a type of chromatography in which the mobile phase is in the state of gas or vapor - an inert gas. It is a carrier gas. The stationary phase is a high molecular weight liquid, which is fixed on a porous carrier or on the walls of a long capillary tube.

GC is a universal method for separating mixtures of substances that evaporate without decomposition. The components of the mixture move through the chromatographic column with a carrier gas. In this case, the mixture is repeatedly distributed between the carrier gas and the stationary phase. The separation occurs due to the different solubility of the mixture components in the solid phase. At the exit of substances from the column, they are recorded using a detector.

The detector is a continuous device that registers an analytical signal. About 60 types of detection systems have been proposed for GC. Table 1.2 lists the types of detectors most commonly used in GCs.

Table 1.2 Gas Chromatography Detectors

Name of the detector	Principle of operation
By thermal conductivity	The difference in thermal conductivity between the analyte and the carrier gas is recorded
Name of the detector	Principle of operation
Electronic gripping	Capture by the analyte of thermal electrons generated by irradiation with β-particles or high-energy electrons of the carrier gas
UV	Absorption of UV light by a specific analyte chromophore
microwave plasma	Excitation of an analyte in a microwave plasma and emission of light at the characteristic wavelength of the element present in the substance
Flame photometric	Excitation of the analyte in a flame and emission of light depending on the type of element present in the substance
Atomic absorption spectrometer	Thermal atomization followed by absorption of light at a specific wavelength
Electrochemical	Absorption of analyzed substances by a liquid flow and their electrochemical detection in the flow
IR spectrometer	Absorption of light in the IR region by the analyte
Flame ionization	Formation and registration of ions in a flame during the combustion of analyzed substances
Mass spectrometer	Formation of molecular and fragment ions by electron impact or chemical ionization
photoionization	Photochemical formation and registration of ions under the action of hard UV radiation on the analyte

The flame ionization detector is sensitive but non-selective to BP. Therefore, it is used only for the analysis of simple mixtures.

The electron-capture detector is both sensitive and selective to BP, but its application is difficult due to the high response to electrophilic compounds.

The use of photoionization is promising for determining BP, but it has not found wide distribution due to the instability of operation and the high cost of equipment.

GC with mass spectrometric detection is the best solution for the determination of benzapyrene in complex mixtures

The principle of operation of a mass spectrometer is to distribute fragments or ions by mass. The process of ionization is used to convert neutral molecules into ions. Electron impact is most often used to ionize organic compounds. In addition, chemical ionization is used, which is based on the occurrence of ion-molecular reactions. To study biological molecules, polymers and other substances that cannot be transferred to the gas phase without decomposition, special types of ionization are used.

GC with mass spectroscopic detection is the only method that allows the use of internal standards for quantitative determinations. Labeled 2 H and 13 C isomeric mixtures of PAHs are used as standard substances. The weight shift and the fact that the reference substances have almost the same characteristics as unlabeled PAHs facilitates identification.

One of the minor disadvantages of mass spectrometers is the small range of linearity of the detector response. Therefore, a conventional mass spectrometer is replaced by a time-of-flight one. Its feature is that in a short time it is possible to obtain a complete mass spectrum of compounds and an accurate measurement of masses up to 0.0001 a.m.u.

The combination of a time-of-flight mass spectrometer with gas chromatography provides good component separation of complex mixtures and low detection limits.

1.2.2 Liquid chromatography

Liquid chromatography (LC) is a method for the separation and analysis of complex substances in which liquid is the mobile phase. On the one hand, the mobile phase performs a transport function, i.e. transfers the non-sorbable substance, and on the other hand regulates the equilibrium constants, and, consequently, retention as a result of interaction with the stationary phase and with the molecules of the substances being separated.

In LC, separations most often take place at room temperature. The analyzed sample is injected into the column and the eluent is passed through. In LC, the nature of the mobile phase is essential. Due to this, various combinations of even a small number of stationary phases and a large number of mobile phases make it possible to solve various analytical problems.

Analysis in classical liquid chromatography is carried out for a long time, since the sample feed rate is low. This method is suitable for preliminary separation of the components of a mixture. In most cases HPLC is used. Fast mass transfer with high separation efficiency allows HPLC to determine molecules, macromolecules and ions. The differences between classical LC and HPLC are shown in Table 1.3.

Table 1.3 Experimental differences between classical and high performance LC

Characteristic	Classical LC	HPLC
Pressure, atm	From fractions of atm. up to 2 atm.
Flow rate, mm/min
Separation duration	From several hours to several days	Several minutes to several hours
Equipment	Speaker and accessories	Chromatograph
Separation type	Preparative separation	Analytical division
Detection	Detection of individual fractions by analytical methods	With a detector
Amount of test substance	From a few micrograms to several kg	Several ng to several micrograms

HPLC is a column chromatography method in which the mobile phase is a liquid moving through a chromatographic column filled with a stationary phase (sorbent). Columns for HPLC are characterized by high hydraulic resistance at the inlet.

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

a) distribution;

b) ion exchange;

c) exclusive;

d) chiral;

e) adsorption.

Partition chromatography is based on the distribution of a substance between two immiscible liquids, similar to repeated extraction. When passing a liquid mobile phase, separation occurs due to the different solubility of the mixture components in the liquid stationary phase.

In ion-exchange chromatography, the molecules of a mixture of substances, dissociated in solution into cations and anions, are separated when moving through the sorbent due to different forces of interaction of the ions being determined with the ionic groups of the sorbent

In size exclusion chromatography, the molecules of substances are separated by size due to their different ability to penetrate into the pores of the stationary phase. In this case, the largest molecules that can penetrate into the minimum number of pores of the stationary phase are the first to leave the column, and the substances with small molecular sizes are the last to leave.

In chiral chromatography, optically active compounds are separated into individual enantiomers (mirror isomers).

In adsorption chromatography, substances are separated due to their different ability to be adsorbed and desorbed from the surface of a sorbent with a developed surface.

Depending on the polarity of the mobile and stationary phases, adsorption chromatography is divided into normal phase (NPC) and reversed phase (RPC) chromatography.

NPC uses a polar adsorbent and a non-polar mobile phase, while RPC uses a non-polar adsorbent and a polar mobile phase.

Although liquid chromatography is a method for separating a sample into components, a modern liquid chromatograph includes not only a separation system, but also a system for quantitatively measuring the content of each component, i.e. detection system. The scheme of the chromatograph is shown in Figure 1.3.

benzapyrene liquid chromatography hydrocarbon

Figure 1.3 Schematic of a liquid chromatograph

1 - pump for supplying the mobile phase through the column

2 - dispenser for introducing the sample into the column

3 - separator column

4 - detector - a device for obtaining an analytical signal

5 - processing system - analytical signal converter into a form convenient for human perception

To register an analytical signal, as mentioned earlier, detectors are used. Various detection methods are used in LC. Some of them are discussed in Table 1.4.

Table 1.4 Detectors in liquid chromatography

Name of the detector	Principle of operation
Spectrophotometric	During the elution process, the optical density of the eluate is measured at a certain wavelength
Fluorimetric	The fluorescent radiation of the absorbed
Name of the detector	Principle of operation
Refractometric	Determination of the concentration of a substance depending on the refractive index different from the refractive index of the mobile phase
Evaporative laser light detector	The principle of operation is based on the difference in vapor pressures of the chromatographic solvents that make up the mobile phase and the analyzed substances

In HPLC, a fluorimetric detector is used to determine BP, which is selective with respect to benzapyrene and has high sensitivity. The principle of operation of such a detector is based on measuring the fluorescent emission of absorbed light. Measurements are mainly carried out in the UV region at the wavelength of maximum absorption for a given group of substances. The wavelength of fluorescent radiation is always greater than the wavelength of absorbed light. Due to the fact that detection is carried out from zero intensity, fluorimetric detectors are more sensitive than absorption detectors.

The combination of HPLC with a mass spectroscopic detector has not been used in the determination of benzapyrene. This is due to the fact that high requirements are imposed on the purity of extracts, i.e. the duration of the analysis increases due to the laborious preparation of the sample. In addition, the equipment is quite expensive and not sufficiently selective and sensitive.

1.3 Determination of benz(a)pyrene in water by HPLC

1.3.1 Measuring instruments, auxiliary equipment, reagents

For measurements, an Agilent 1200 HPLC chromatograph (Fig. 1.4) with a fluorimetric detector is used, which provides an excitation wavelength range in the range of 270-365 nm and fluorescence registration in the range of 390-460 nm.

Figure 1.4 Agilent 1200 HPLC

The chromatographic column is filled with a sorbent for OFC. Under the conditions of the experiment, the efficiency of the column should be at least 5000 theoretical plates.

For sample preparation, a separating funnel with a capacity of 2000 cm 3, a rotary evaporator, a water bath, a water jet pump, n-hexane, sodium chloride and sulfate, and acetonitrile are used.

To prepare calibration solutions, flasks with a capacity of 25 and 50 cm 3 and graduated pipettes with a capacity of 1, 2, 5 cm 3, a solution of benz(a)pyrene with a concentration of c = 1.0 μg/cm 3 and acetonitrile are used.

1.3.2 Sample preparation

Liquid-liquid extraction (LLE) is used to extract benzo(a)pyrene from water. Extract the benzo(a)pyrene with n-hexane. For this purpose, selected water with a volume of 1000 cm 3 is introduced into a separating funnel with a capacity of 2000 cm 3 and 25-30 cm 3 of n-hexane and 20 cm 3 of sodium chloride (NaCl) with a concentration of c=0, 25 g/cm 3 are added and extraction is carried out, shaking the mixture for 10-15 minutes. After that, the aqueous layer is separated and extraction is carried out twice more without adding NaCl. After the extracts are combined and dried, passing through a desiccant, which is a funnel with a layer of sodium sulfate at least 2 cm high. Dichloromethane of the same volume as n-hexane can be used as an extractant.

After extraction, the extract is evaporated to a volume of 3 - 5 cm 3 on a rotary evaporator - a device for the rapid removal of liquids by distillation under reduced pressure. The residue is transferred into a test tube with a capacity of 10 - 15 cm 3 and n-hexane is added, after which the solution is evaporated to dryness in a vacuum of a water jet pump, placing the test tube in a water bath at a temperature of 40 - 50 ° C. The residue is dissolved in 0.2 - 0.5 cm 3 acetonitrile. The resulting concentrate is kept for at least 15 minutes.

1.3.3 Graduation

Graduation is carried out according to 5 standard solutions with a concentration of 0.002; 0.01; 0.02; 0.05 and 0.1 µg/cm3. The preparation of calibration solutions is described in Table 1.5.

Table 1.5 Preparation of calibration solutions

	s, µg/cm3	c0, µg/cm3

The solutions were made up to the mark with acetonitrile.

At least two chromatograms are recorded for each of the solutions. Figure 1.5 shows a chromatogram of benzo(a)pyrene with a concentration of 0.002 µg/cm 3 .

Figure 1.5 Chromatogram of benz (a) pyrene with a concentration of 0.002 μg / cm 3

The discrepancy between the obtained areas should be no more than 7% of their arithmetic mean.

Based on the data obtained, a calibration graph is built (Fig. 1.6) in the form of a dependence of the peak area on the mass concentration. The calibration curve must be linear. For each solution, the deviation of the mass concentration measured according to the calibration characteristic from the specified value should not exceed 12%. If the deviation exceeds the specified value, then the calibration is repeated. Calibration is carried out not only before measurements, but also after replacing the column or during maintenance work of the chromatograph.

Figure 1.6 Calibration dependence of the peak area on the concentration of benzo(a)pyrene (r 2 = 0.998)

1.3.4 Performing HPLC analysis

Chromatographic determination of benzapyrene is carried out in an Agilent 1200 HPLC column, the column efficiency should be at least 5000 theoretical plates based on the benzapyrene peak. The inner diameter of the column is 2 mm. The column is filled with a reversed-phase sorbent (RPS). In this definition, a pre-column was used that performs a protective function. The inner diameter of the pre-column is 2 mm; it is filled with the same OFS.

As OFS, sorbents with bonded phases obtained on the basis of silica gel are used. In this definition, octadecyl silica gel (C 18) with a particle size of 5 μm and hydrophobic microporous hypercrosslinked polystyrene with a particle diameter of 3.2 μm and an average pore diameter of 20–40 E were used as sorbents. This sorbent is chemically stable at pH = 2–7. Separation efficiency is ensured by the high surface area of ​​the sorbent particles, as well as the uniformity of the sorbent composition and dense uniform packing. The capacity factor (k) of such a sorbent is 9.86.

The mobile phase is a mixture of acetonitrile - water. Prepare the eluent in a volume ratio of 8:2. In glassware with a capacity of 1000 cm 3 add 200 cm 3 of water and bring to the mark with acetonitrile. Immediately before use, the eluent is kept for degassing for at least 4 hours. For faster degassing, the container with the eluent is evacuated by connecting it to a water jet pump and placing it in an ultrasonic bath. The eluent is supplied by a loop dosing valve (injector) with a loop volume of 10 mm 3 , the flow rate of the mobile phase is 200 mm 3 /min.

Under these conditions, chromatography takes 20-30 minutes.

1.3.5 Registration and processing of results

Benzopyrene is detected using a fluorescent detector. It is recommended to register the chromatogram at the excitation wavelength l ex = 365 nm and the registration wavelength l register. = 400 - 460 nm.

To increase the sensitivity in this technique, the wavelengths of excitation and emission by fluorescence were programmed. Programming mode is shown in table 1. 6.

Table 1.6 Programming mode when using a fluorescent detector

The chromatogram obtained from water analysis is shown in Figure 1.7.

Figure 1.7 Chromatogram of BP content in water

The peak of benzapyrene is determined by the retention time, because this is a qualitative characteristic of benzo(a)pyrene. To quantify the content of benzo(a)pyrene, the peak area is calculated. Then, according to the calibration graph, its concentration is found.

If the concentration of benzo(a)pyrene exceeds the maximum concentration of the standard solution, then the analyzed solution is diluted and the sample is analyzed again.

The sensitivity of the determination of benzapyrene by this method is 0.01 µg/DM 3 .

1.4 Determination of benz(a)pyrene in water by GC

1.4.1 Sampling and preparation

An important part of the analysis is the selection and preparation of the sample. Water is taken several times with an interval of several days. After that, the water is filtered and a sample of 0.5 liters is taken. Sodium chloride is added to the sample and stored in the refrigerator for no more than a day.

To obtain reliable data, BP is extracted from the sample by the LLE method. The extractant is diethyl ether. Extraction is carried out three times. The first two times the substance is extracted in a volume of 50 ml of the extractant. For the third time, the volume of the extractant is 30 ml. All extracts are combined and evaporated in a rotary evaporator until the ether is completely removed. The resulting sample is dissolved in 2 ml of benzene.

Before quantitative determination of BP, the components of the mixture are separated by thin layer chromatography (TLC).

TLC is a chromatographic method based on the use of a thin layer of adsorbent as a stationary phase.

Before separation, the plate is immersed in a 4% solution of caffeine in chloroform and activated in an oven at a temperature of 100 ° C. To remove impurities from the plate, after cooling it, it is washed with chloroform and activated again in an oven for 30 minutes at a temperature of 100 about S.

Samples dissolved in benzene are placed on a plate and chromatographed. The eluting agent is a mixture of cyclohexane - n-hexane, in a volume ratio of 16:1.

1.4.2 Quantification

Quantitative analysis according to this method is carried out by gas-liquid chromatography with a flame ionization detector. For analysis, a chromatograph "Tsvet - 500" (Fig. 1.8) with a flame ionization detector was used.

Figure 1.8 Gas chromatograph "Color - 500"

The carrier gas for this determination was nitrogen. Nitrogen was supplied at a flow rate of 3 ml/min. A capillary quartz column with a size of 25 m × 0.32 mm was used for analysis. Methyl silicone oil OV-101 with a film thickness of 0.4 µm was used as the stationary phase. The analysis was carried out in the temperature programming mode from 210 to 300°C at a rate of 4°C/min. A 1 μL sample was injected using a microsyringe.

Figure 1.9 shows the BP chromatogram obtained by this method.

Figure 1.9 Chromatogram of BP obtained by GC (3-BP)

Qualitatively, BP was determined by the retention time, compared with the retention time of the standard sample, which was 28 minutes.

Quantitatively, BP is determined by the external standard method. To do this, build a calibration graph for several standard samples with a concentration range of 1-20 ng/ml. The peak height was used as an analytical signal.

Benz (a) pyrene is a carcinogen of the 1st hazard class related to PAHs. It can be found in soil, water, air, food, and when it enters the body, it accumulates. Therefore, its determination in various objects is a priority.

MPC for BP is quite low, so sensitive methods are used to determine it. The difficulty of its determination also lies in the fact that, in addition to BP, the sample may contain its isomers, such as benz(e)pyrene and perylene, whose retention time is almost the same as that of BP. That is, the presence of isomers makes it difficult to identify the substance. This problem is solved by first separating the mixture by TLC, as well as using standard BP samples.

In this course work, several chromatographic methods were considered that are suitable for the determination of benzo (a) pyrene. After a preliminary work with the literature, the choice of methods for its quantitative determination was justified. On the basis of the method, the methods were selected and the chromatograms obtained by these methods are shown.

BIBLIOGRAPHY

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3. Pat. 2466406 Russian Federation. Methods for the quantitative determination of benzo (a) pyrene in urine by liquid chromatography / Zaitseva N. V., Ulanova T. S., Kornazhitskaya T. D., Kislitsina A. V., Pshenichnikova E. O., Permyakova T. S. - - No. 2011142553/15; dec. 20.10.11; publ. 10.11.12, Bull. No. 31.

4. Maximum allowable concentrations (MPC) of chemicals: GN 2.1.7.2041-06. -- [Introduced 2006-02-07]. - M.: Standartinform, 2006. - 7 p.

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Benzopyrene belongs to the class of polycyclic aromatic hydrocarbons - PAHs. This is a group of organic compounds in the chemical structure of which there are benzene rings - groups of three rings or more. Chemical definition of benzapyrene: an organic substance containing carbon, which is part of the group of polycyclic hydrocarbons, with molar mass 252.31 g/mol.

What is benzapyrene

Benzopyrene, like all PAHs, is mainly the result of technological progress, a consequence of human activity. The main sources of technogenic pollution of PAHs are the combustion of solid and liquid organic substances, including oil and oil products, wood, and anthropogenic waste. Of the natural sources of benzapyrene, it is worth noting forest fires, volcanic eruptions.

However, the formation of benzapyrene can also occur without combustion processes - during pyrolysis, smoldering, polymerization.

Benzapyrene is released during smoking: the content of benzapyrene in the smoke of one cigarette averages 0.025 mcg, which is many times higher than the MPC (10,000-15,000 times on average). It has been calculated that smoking one cigarette is equivalent to sixteen hours of exhaust gas inhalation in terms of its benzapyrene content.

Benzopyrene formula

There are two isomers of benzapyrene. The first is 1,2-benzapyrene (3,4-benzpyrene) - contained in all combustion products - oil, tar, coal, smoke of various origins, including. In its pure form, these are needle-shaped crystals or plates of light yellow color, with a melting point of about 177 ° C.

4,5-Benzopyrene - crystals in the form of needles and plates of light yellow color, with a melting point of 179 ° C. Contained in coal tar, found in soils (especially near enterprises and highways). It does not have mutagenic or carcinogenic properties.

The chemical formula of benzopyrenes is C20H12.

Benzopyrene in soil and air

Benzopyrene practically does not occur in the free state, but always precipitates on particles contained in the air. Together with moving air masses, benzapyrene spreads over a large area, and falling out of the air together with solid particles (for example, during precipitation) enters the soil layers, reservoirs, and on the surfaces of buildings.

In the migration and accumulation of benzapyrene, such a source as road transport also plays a role. On the one hand, moving over long distances, cars contribute to the uniform distribution of benzapyrene. On the other hand, settled benzapyrene accumulates in large quantities along highways and on objects next to them (the so-called "secondary sources").

Benzopyrene is easily “included” in the cycle of substances in nature: with precipitation, which always contains solid particles, it is carried even to territories remote from the main source of PAHs, enters water bodies, from where, during evaporation processes, it rises again into the air. It is this ability of benzapyrene to migrate that causes its content to be high in places where there is no powerful source of this substance.

Getting into the environment and accumulating in it, benzapyrene penetrates into plants, which later serve as livestock feed or are used in human nutrition. The concentration of benzapyrene in plants is higher than its content in the soil, and in food (or feed) is higher than in the feedstock for their manufacture. This effect of increasing the concentration of chemicals, including benzapyrene, is called bioaccumulation.

Thus, benzapyrene is dangerous not only as a background pollution of the environment, but also as a substance penetrating the body through the food chain.

MAC of benzapyrene

The main method for the determination and control of benzapyrene is liquid chromatography.

According to the Hygienic Standards 2.1.6.695-98 and 2.1.6.1338-03, the maximum allowable average daily amount of benzapyrene in the air (MPCds) is 0.1 μg/100 m3 or 10-9 g/m3, and its MPC in soil according to the Hygienic Standards 2.1. 7.2041-06 - 0.02 mg / kg in total, taking into account the background level. In the air at workplaces, the average shift MPC is not more than 0.00015 mg / m3. (from clause 1. and clause 2. GN 2.2.5. 1313-03).

MPC of benzapyrene in water is not more than 0.000001 mg/l, in drinking water with a centralized water supply system - not more than 0.000005 mg/l. In bottled drinking water - from no more than 0.001 µg/l (water of the highest quality) to no more than 0.005 µg/l in bottled water of the first quality category.

In food products in which the presence of benzapyrene is permissible due to technological features, the permissible level of benzapyrene is not more than 0.001 mg / kg. These include: sausages and products using by-products, including smoked ones; smoked lard; sausages and smoked products from poultry meat and offal; smoked canned food and fish preserves, smoked fish; food grain.

When using smoke flavorings, the content of benzapyrene is not more than 2 µg/kg (l), and after their use, the content of benzapyrene in finished products should not exceed 0.03 µg/kg (l).

In other food products, the presence of benzapyrene is not allowed.

However, according to the results of monitoring, the norms for the content of benzapyrene are exceeded many times over. On average, the level of air pollution in cities is 5-12 times higher than the MPC, in soil - 3-7 times, in food - from 1.5 to 11 times.

The effect of benzapyrene on the human body

Benzopyrene is classified as a substance of the first hazard class. The first hazard class is substances with an extremely high hazardous impact on the environment, while the changes caused by them are irreversible and cannot be restored.

Benzopyrene is one of the most powerful yet widespread carcinogens. Being chemically and thermally stable, possessing the properties of bioaccumulation, once it enters and accumulates in the body, it acts constantly and powerfully. In addition to being carcinogenic, benzapyrene has a mutagenic, embryotoxic, and hematotoxic effect.

The routes of penetration of benzapyrene into the body are varied: with food and water, through the skin and by inhalation. The degree of danger is regardless of how the benzapyrene entered the body. In experiments, as well as according to monitoring of environmentally unfavorable areas, benzapyrene is introduced into the DNA complex, causing irreversible mutations that pass into subsequent generations. Of particular concern is the fact of bioaccumulation of benzapyrene: the likelihood of developing mutations in the next generations of offspring increases many times due to bioaccumulation.

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