Method for determination of residual quantities of pesticides in milk and meat

 

(57) Abstract:

Use: in analytical chemistry, in particular in methods for determining pesticides in agricultural products and environmental objects thin-layer chromatography, and can be used to control content in milk and meat N, n-dicyclohexylamine sorbic acid (carbamide), part of the preparations "Darwen" and "Denavas". Entity: definition of micro carbamide are on standard plates with a thin layer of sorbent fixed starch in the mobile phase consisting of a mixture of carbon tetrachloride and acetone in a volume ratio (20 15) : 1, with subsequent detection zone localization analyte processing chromatograms reagent Dragendorff modified with 0.2% aqueous solution of ascorbic acid.

The invention relates to analytical chemistry of pesticides, in particular to methods for determination of pesticides in agricultural products and environmental objects by means of thin layer chromatography, and can be used to control content in milk and meat N,N-dicyclohexylamine sorbic acid (hereinafter referred to as carbamide), the N,N-disubstituted amides of carboxylic acids using the method of thin-layer chromatography on plates with a thin layer of silica gel or aluminium oxide, fixed in calcium sulfate or gypsum and painted by hand on glass plates [1-3]

So, there is a method of determining in the air of working zone of pesticide diphenamid containing N, N-disubstituted amide group, using the method of thin-layer chromatography in thin layer of aluminum oxide, enshrined in calcium sulfate and painted by hand on a glass plate, in a mobile phase consisting of a mixture of hexane, acetone and ammonia in the volume ratio of 10: 5: 0.8 seconds subsequent processing of chromatograms reagent Dragendorff. The detection limit of 1 ug [2]

The disadvantage of this method is the use of non-standard chromatographic plates with a thin layer of sorbent applied manually, which reduces the accuracy, sensitivity, selectivity determine due to the uneven thickness of the layer of sorbent.

Usually applied in the analysis of standard chromatographic plate (Silufol, Alohol, records of the Armenian branch of area "Armor" and others) contain as binder starch. The handling of such records reagent Dragendorff and various modifications [4 and 5] leads to the fact that the background plate darkens due to the impact on starch free iodine, you is asego in the composition of the reagent. This fact hinders the identification of spots of the analyte in the chromatogram as orange spots are masked by the dark background of the plate. Therefore, the definition of substances both qualitative and quantitative on such plates using reagent Dragendorff difficult.

Closest to the proposed technical essence is a method for determining residues of the pesticide in milk [6] involving the extraction of pesticides from the analyzed samples and the obtained lipid balance, purification, and concentration of the extracts and their subsequent elution movable solvent, applying the sample on a plate with a layer of sorbent, the chromatography was carried out, the manifestation of the areas of localization of the desired component (the residual amount of the pesticide) by processing the chromatograms reagent with subsequent quantification of the desired component on the results of a visual comparison of the intensity of the color and size of the spots of the sample with the same characteristics of the spots of the standard solutions.

The disadvantage of this method is the impossibility of determining trace amounts of carbamide, part of the drug "Darwen" and "Denavas" in the study kachestve to resolve the defect by that fixing a thin layer of sorbent on chromatographic plates in the mobile phase is performed by the starch, as mobile phase a mixture of carbon tetrachloride with acetone in a volume ratio (20-15):1, and to detect areas of localization of carbamide on chromatographic plates from a number of reagents choose the reagent Dragendorff modified with 0.2% aqueous solution of ascorbic acid.

For chromatographic separation of the sample is used as a chromatographic sorbent plates "Silufol", plate "Armor" Armenian branch of area and other standard plate with a thin layer of silica gel, fixed starch, and as a chromogenic reagent modified reagent Dragendorff containing, in contrast to the above formulations, ascorbic acid, sulfite or sodium bisulfite, which due to its restorative properties associated free iodine, so that the background plate becomes light yellow. The use of ascorbic acid is preferred as the background color of the plate remains stable for a longer time than when using sulfite and sodium bisulfite.

For and in milk and meat, the analyzed sample pre-extracted with the appropriate solvent, the extract was subjected to purification and concentration, and then conduct a quantitative definition as follows.

Standard chromatographic plate with a thin layer of sorbent fixed starch, at a distance of 15 mm from the edge outline the starting line and at its middle with microspace put the extract of the sample, and the right and left of the spot samples at a distance of 15 mm from one another cause standard spot-witnesses with different content of the analyte. The diameter of the marked spots should not exceed 10 mm, the number of spots on the disc, 7-9.

The method is as follows.

The plate is placed in a closed polished glass plate chromatographic chamber size HH cm, filled for 30 min prior to chromatography was carried out (to saturate the chamber with solvent vapors) mobile phase consisting of a mixture of carbon tetrachloride with acetone (all components of the qualification of "chemically pure" or "analytical grade"). The optimum volumetric ratio of carbon tetrachloride and acetone (20-15):1. The increase or decrease of this ratio leads to erosion pastwo mobile phase in the chromatographic chamber should be such, to the chromatographic plate was immersed in the mobile phase is not more than 0.5, see Plate elute a bottom-up fashion, the height of elevation of the front of the mobile phase 10 see the duration of the elution 15-20 minutes Then the plate is removed from the chamber, dried in air 5-8 min and treated with spray reagent Dragendorff modified aqueous solution of ascorbic acid.

Modified reagent Dragendorff obtained by dissolution of 850 mg of the basic bismuth nitrate in 40 ml of water and 10 ml of acetic acid. To this solution was added 8 g iodotope potassium, dissolved in 20 ml of water. For processing chromatogram 1 ml of the obtained solution was diluted with 2 ml of acetic acid and 10 ml of 0.2% aqueous solution of ascorbic acid. The specified concentration of aqueous ascorbic acid solution is optimal. The decrease in the concentration of an aqueous solution of ascorbic acid leads to a rapid darkening of the background plate, and an increase in the concentration does not increase the effectiveness of ascorbic acid.

After processing the chromatogram reagent Dragendorff modified ascorbic acid, manifested spot carbamide orange color with sootvetstvuyscee and square spots of the analyte and the standard, or gauge dependence of the spot area (mm2from the amount (μg) of a substance, or by means of a densitometer according to the ratio of the integral values of the substance content in the spots of the sample and standard.

P R I m e R 1. In a volumetric flask to prepare a solution of carbamide in acetone concentration 0.05 mg/ml

In the middle of the start line chromatographic plates "Silufol is applied in the form of spot 10 ál of this solution, which corresponds to the content of carbamide in the spot of 0.5 μg. On both sides of the spot sample is applied in the form of separate spots at a distance of 15 mm from each other by 10 ál of the standard solutions of concentrations carbamide 0,05; 0,10; 0,20; 0,40; 0,80; 1,0 mg/ml Plate elute in the mobile phase carbon tetrachloride + acetone (15:1) at a height of 10 cm, and then treated with spray reagent Dragendorff modified ascorbic acid. On the chromatogram appear clear orange spot carbamide on a light yellow background with Rf0,42. Via a calibration curve, constructed by the dependence of the square of the standard spots from the content of carbamide, determined in spot samples of 0.52 μg. The relative error of determination is 5%

P R I m m e R 2. Determination carried out analogously to example 1, I the background color after 6-7 min darkens.

P R I m e R 3. Determination carried out analogously to example 1, but for modifying reagent Dragendorff use of 0.4% aqueous solution of ascorbic acid. The result of the determination coincides with the result of example 1.

P R I m e R 4. Determination carried out analogously to example 1, but on the chromatographic plate put 10 μl of an acetone solution of carbamide a concentration of 0.5 µg/ml, which corresponds to the content of carbamide on spot 5 µg. Found a 5.25 µg. The value of Rf0,43. The relative error of determination 5%

P R I m e R 5. Determination carried out analogously to example 1, but on the chromatographic plate put 10 μl of an acetone solution of carbamide a concentration of 1 mg/ml, which corresponds to the content of carbamide in spot 10 ál. Found a 9.5 µg. The relative error of detection of 5% of the Value of Rf0,44.

P R I m e R 6. Determination carried out analogously to example 1, but the volume ratio of the components of the mobile phase carbon tetrachloride + acetone 20: 1. The value of Rfcarbamide 0,42. The detection limit of 0.5 μg. The relative error of determination 5%

P R I m e R 7. Determination carried out analogously to example 1, but the volume ratio of the components of the mobile phase carbon tetrachloride, asmitou. The relative error of determination of 9.8%

P R I m e R 8. Determination carried out analogously to example 1, but the volume ratio of the components of the mobile phase carbon tetrachloride + acetone 30:1. The value of Rfcarbamide 0,35. The detection limit of 1 µg. The fuzzy spot. The relative error of determination of 10.2%

P R I m e R 9. Determination carried out analogously to example 1, but for processing chromatograms using the reagent Dragendorff without modification of the aqueous solution of ascorbic acid. Spot carbamide on the chromatogram is masked by the dark blue background. Detection is impossible.

P R I m e R 10. Determination carried out analogously to example 1, but using chromatographic plate "Armor". The value of Rffor carbamide of 0.87. The detection limit of 0.5 μg. The relative error of determination 5%

P R I m e R 11. 200 ml of milk containing carbamide 0.005 mg/l is poured into 300 ml of acetone, incubated for 15 min, then filtered under vacuum, the residue on the filter is washed with 20 ml of acetone. The filtrate is transferred into a flask with a glass stopper, contribute 40 g of sodium chloride, close the tube and intensively shaken until the appearance of the emulsion (1-3 min). The emulsion is poured into a separating funnel and extragere wodnego sodium sulfate (80 g), evaporated to odorless solvent. The oily residue is dissolved in 1-2 ml of hexane and transferred to a column of aluminum oxide, obtiva flask 2-3 times the same portions of hexane. Hexane sucked off under vacuum, the column elute with a speed of 80 to 100 drops per minute first 30 ml of hexane (eluate discarded) and then with a mixture (10 ml), hexane + acetone in a volume ratio of 50:1. This eluate evaporated to dryness. The residue is dissolved in 2 ml of hexane and then evaporated in a weak current of air to volume of 0.2-0.3 ml of This volume using microspace or glass capillary is applied to the chromatographic plate "Silufol" and spend the definition analogously to example 1. The value of Rfcarbamide 0,43. In the analyzed sample is detected 0.004 mg/l carbamide. The total relative error in the determination of 20% of

P R I m e R 12. 40 g of ground beef containing carbamide 0.05 mg/kg extracted twice with a mixture of 60 ml of acetone with 10 ml of water by shaking each time for 30 min, and filtered under vacuum. The filter residue is washed with 20 ml of acetone. In the combined filtrate contribute 30 g of sodium chloride and intensively shaken until the appearance of the emulsion. The emulsion is poured into a separating funnel and extracted with hexane three portions 60 is to odorless solvent and then proceed as in example 11. The value of Rfcarbamide 0,43. The amount of substance, defined in the sample, consistent with the content of carbamide 0.04 mg/kg total relative error of determination 20%

P R I m e p 13. Determination carried out analogously to example 11, but samples of milk contains 0.002 mg/kg carbamide. Found a spot carbamide not quantifiable.

Thus, this method allows you to:

to determine carbamide milk and meat;

to extend the analysis capability carbamide by thin layer chromatography through the use of along with custom plates with a thin layer of sorbent enshrined in calcium sulfate or gypsum, standard chromatographic plates with a thin layer of sorbent fixed starch;

to increase the selectivity of the analysis by using as mobile phase a mixture of carbon tetrachloride and acetone in a volume ratio (20-15): 1, which allows to obtain a clear spot carbamide, as well as to separate the spot carbamide from interfering coextraction substances extracted during the extraction of milk and meat;

to increase accuracy through the use of standard chromatographic plates;

to increase the thief of ascorbic acid, to lighten the background on the chromatogram.

METHOD for determination of RESIDUAL QUANTITIES of PESTICIDES IN MILK AND MEAT, providing for the extraction of pesticides from the analyzed samples and isolated from her fat balance, purification, and concentration of the extracts and their subsequent elution movable solvent, applying the sample on a plaque mounted on it with a layer of sorbent, the chromatography was carried out, the manifestation of the areas of localization of the desired component by processing the chromatograms reagent with subsequent quantification of the desired component on the results of a visual comparison of the intensity of the color and size of the spots of the sample with the same characteristics of the spots from the standard solution, characterized in that when determining residues of pesticide establish microquantities of carbamide, part of the preparations Darwen and dichaves, when this pin to plate a layer of sorbent carry out the starch in the mobile phase, use a mixture of carbon tetrachloride with acetone in a volume ratio 20 15 1, and to detect areas of localization of carbamide on chromatographic plates from a number of reagents choose the reagent Dragendorff, modificirowan the

 

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