SU1644880A1 - Method for quantitative determination of benzoic and sorbic acids in foods and beverages - Google Patents

Description

one

(21) 4612582/13 (22) 05.12.88 (46) 04/30/91. Bul Number 16

(71) Institute of Nutrition, Academy of Medical Sciences of the USSR

(72) D. B. Melamed, B. G. L. pkov and T.V. Kiseleva

(53) 543.544: 637.07 (088.8)

(56) Melamed D. B. and others. Analysis of food additives. Preservatives (review). Honey. ref. J., 1987, sect. VII, n ° 8, p. 28-34.

J. Yosseleetc. Thin Layer chromatcgraphy separation of preservatives. J. Chromatogr. 1966, v. 23, N 2, p. 305-308.

(54) METHOD FOR QUANTITATIVE DETERMINATION OF BENZOIC AND SORBIC ACIDS IN FOOD AND DRINKS

(57) The invention relates to the food industry and can be used to determine benzoic (BC) and sorbic (SC) acids in food and beverages. - The purpose of the invention is to increase the reliability of the results and simplify the process by providing a better separation of the BC and IC spots. A sample containing BK and SC is extracted from a sample of a food or drink, and the points of the obtained sample and standard solutions containing a mixture of BK and SC are applied on a chromatographic plate. Then one point of the sample and one point of the standard solution are treated

oxidative mixture (OS) containing formic acid and hydrogen peroxide in a 1: 1 ratio. The plate is dried, heated at 80-100 ° C for 15-20 minutes, allowed to cool, and TLC in a solvent system is carried out with petroleum ether — chloroform — formic acid — diethyl ether 10: 4: 1: (0.8 1.6). The identification of spots of BK and SC on the plate is carried out in UV light with A 254 nm, BK, when treated with an oxidizing mixture (OS), is converted to n-hydroxybenzoic acid, Rf of which is approximately the second less than Rf BK. SK when processing the OS forms malonic aldehyde, not absorbing UV light with A 254 nm. It is identified by spraying the plate with a 2-thiobarbituric acid solution, resulting in a pink spot. Untreated points give dark spots in UV light with corresponding Rf. The points of the sample, standard solutions and blank samples (ethyl acetate) are applied to the second plate. Next, the points on the plate are treated as described above, and TLC is carried out, the BC and SC spots are identified, marked, cut out and eluted with ethyl acetate. The optical density of the eluates was measured on a spectrophotometer at A 272 and 254 nm for Bq and CK, respectively, and the number of Bq and CK in the sample is calculated. 1 dw., 2 tab.

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The invention relates to the food industry and can be used to determine benzoic and sorbic acids in food and beverages.

The purpose of the invention is to increase the reliability of the results and simplify the process by providing a better separation of the benzoic and sorbic acid spots.

The method for quantitative determination of benzoic (BC) and sorbic (SC) acids in food and beverages is as follows.

A sample containing BK and SC is separated from a sample of a food or drink; one of the known methods, for example, is homogenized with sodium sulfate and sulfuric acid, and BK and SC are evaporated with steam into a receiver containing alkali. The distillate is acidified to pH 2-3, saturated with sodium sulfate and extracted with an organic solvent (diethyl ether, ethyl acetate). The extract is dried with calcined sodium sulphate. An aliquot of the extract is concentrated and several points are plotted on a plate with Silufol UV 254. Dots of different volumes of a standard solution containing mixtures of BC and SC are next to each other. In one of the stains of the sample and standards, the mixture is treated with a mixture of formic acid and hydrogen peroxide in a 1: 1 ratio, dried, and the plate is placed in a drying oven heated to 80–90 ° C for 15–20 min. It is then allowed to cool and immersed in a thin layer chromatography (TLC) chamber containing a solvent system of petroleum ether-chloroform-formic acid-diethyl ether 10: 4: 1: (0.8-1.6). To identify the chromatogram is examined in UV light with A 254 nm. The presence of a dark spot on the chromatogram of the untreated point of the sample, whose Rr corresponds to the spot of the SC, and the absence of such a spot on the chromatogram of the treated point of the sample indicates the presence of a spot in the sample. The presence of a dark spot on the chromatogram of an untreated sample point, Rf of which corresponds to the stain of BC, its disappearance on the chromatogram of the treated point of the sample and the appearance on this chromatogram of a spot with Rf approximately three times smaller than the Rf of BC, proves the presence of BC in the sample. Based on the obtained chromatogram, the approximate content of BK and SC in the sample is estimated by comparing the intensity of the absorption of UV light in the sample and standards. An aliquot of the concentrated extract and standard solution was applied to a new Silufol UV 254 plate. so that the numbers of BC and SC standards are close to their estimated content in the extract. Nearly applied to a blank sample in a volume equal to the aliquot of the extract. The blank sample is prepared by soaking up ethyl acetate, similar to the sample extract. In order not to overload the thin layer of sorbent extract, a solution of standards and a blank sample are applied in several adjacent points. The plate is chromatographed under the same conditions as the previous one, dried and examined in UV light with I 254 nm. Mark dark spots of BC and SC on the chromatogram of the extract

and standards, as well as the corresponding Rf chromatographic zones of the blank, cut them out and elute with ethyl acetate. The spectrophotometer measures the optical density of the BA eluates and the IC of the chromatogram of the extract relative to the eluates of the corresponding chromatographic zones of the blank sample, and the eluates of the standards are relative to ethyl acetate, at appropriate wavelengths (272 and 250 nm). If the concentration of BC and / or SC in the sample is too high, the eluates should be diluted with ethyl acetate before measuring the optical density.

The content of BC and IC in the product or

drink calculated by the formula

 K FH0 Y m

((mg / kgili mg / l).

No -v-t-ivr

where K is a coefficient taking into account the completeness of extraction, equal for benzoic acid 1.2, for sorption acid 1.25;

F is the dilution factor of the eluate before measuring the optical density;

The HQ optical density of the experimental probe would be relatively idle, sd. ed ..

No — optical standard for ethyl acetate, standard units,

Y is the volume of the extract, ml;

V is the volume of the extract deposited on plate 0, μl;

t is the concentration ratio of the extract (equal to 10 when the fraction of the extract is evaporated 10 times);

M is the mass of the product, taken for analysis, g;

m is the number of standard deposited on the plate, µg.

The drawing shows the chromatogram of the sample (points 3, 4, 5) and the standard without 0 (points 2 and 6) and after (point 1) the treatment with formic acid with hydrogen peroxide.

In tab. Figure 1 shows the results of separation of BC and SC at 22 ° C in the tested systems of 5 solvents obtained by introducing different volumes of diethyl ether into the petroleum ether – chloroform formic acid 10: 4: 1 solvent system.

As can be seen from the table. 1. introduction of 0.8–1.6, predominantly 1.0–1.2 parts by volume of diethyl ether into the starting solvent system, makes it possible to clearly separate the BC and SC spots, while the best difference between the lower limit The spot of BC 5 and upper SC (column ARr 100 in Table 1) is observed in systems with a content of 1 and 1.2 ob.h. diethyl ether. When the amount of ether is greater than 1.6 and less than 0.8 vol., The spots in the mixture are practically not divided.

Under the action of the BA of a mixture of formic acid with hydrogen peroxide, n-hydroxybenzoic acid is formed, Rf of which is approximately three times less than Rf of BA.

Under the action of this mixture on the SC spot, malonic aldehyde is formed, which does not absorb UV light with A 254 nm.

In tab. Figure 2 shows the effect of the temperature and duration of heating the spent plate mixture on the reaction of BC and SC with a mixture of formic acid and hydrogen peroxide.

Example Definition of BC and CK in block marmalade.

10.5 g marmalade is weighed, homogenized in a mortar with 40 g N32S04 and 40 ml of 1 M H2SO4 and distilled with steam to collect about 120 ml of distillate and a receiver containing 10 ml of 1 M NaOH. Half of the distillate is acidified with 1 M H2S04 to pH 2-3,

saturated with 30 g of NaaSO / i and extracted 3 times with 10 ml of ethyl acetate. The combined extract is dried with 2 g of Na2SO4. 15 mg of the extract are evaporated to 1.5 ml.

A standard solution is prepared as follows: 100 mg of benzoic acid and 10 mg of sorbic acid are transferred to a 50 ml volumetric flask and made up to the mark with ethyl acetate; the concentration of benzoic acid in the resulting solution was 2.0 mg / ml, the sorbic acid was 0.2 mg / ml.

The plate is filled with UV254 15 x 15 cm forcefol (see the chromatogram) at points 1.2 and 6 for 1.1 and 4 µl of the standard solution, while the amount of BK in the spots is 2.2 and 8 µg, SC 0,2,0,2 and 0,8 mcg, respectively. At points 3, 4 and 5, 3.3 and 10 µl extract each was added. Points 1 and 3 are treated with 4 µl of a freshly prepared mixture of equal volumes of formic acid and hydrogen peroxide, dried with warm air and placed in a drying oven heated to 80 ° C for 20 minutes, allowed to cool and immersed in a TLC chamber containing a solvent system: petroleum epr-chloroform-formic acid-diethyl ether 10: 4: 1: 1,2. The plate is examined in UV light with A 254 nm. On the chromatograms obtained from points 4 and 5, the spots are clearly visible, according to the value of Rf, the corresponding BC and SC. The spots from point 5 are about three times more intense than from points 4. At the same time, chromatograms obtained from points 1 and 3 show no spots with Rf BC and SC, but there was a spot with Rf approximately three times smaller than Rf BC . This indicates the presence in the sample of BC and SC, Ptn BK and SC on chromatograms are well separated from impurities and separated. They compare the intensity of the absorption of UV light from the standards and sample and estimate the content of BK and SC in the sample spots. The outcome of this assessment, on the plate is Silufol UV 254

15 x 15 cm applied 60 µl of sample. 30 μl of standard solution and 60 μl of blank (15 ml of ethyl acetate, evaporated to 1.5 ml), the plate is placed in the same solvent system, chromatographed, dried, cut out the zones corresponding to the BC and SC in the experimental, blank samples and standards, placed pour them into test tubes, pour 3 ml of ethyl acetate, cover with lids for three days 1-2 minutes and measure

the optical density of the eluants of the test sample is relatively idle, and the standards are relative to ethyl acetate on a spectrophotometer in cuvettes with I 10 mm at A 272 and 250 nm for BC and SC, respectively. With

measuring the optical density of the SC eluate from the test sample and the standard, the absorption intensity is very high (off scale of the instrument), therefore it is diluted with ethyl acetate 10 times (also diluted

corresponding blank test).

The calculation carried out by the formula:

SBC 2000 K F, N °. u. "MM ° 2000 -1.2 x

x 1

No -v-t-M

0.170 -15-60

0.185 60 10 10.5

 315mg / kg;

35

SSK 2000 1.25 10 x

0.105 -15-6

 242 mg / kg;

0.155 / 60 10 -.10.5 showed that in the tested block marmalade 315 mg / kg benzoic and 242 mg / kg

sorbic acid.

PRI mme R 2. Determination of benzoic and sorbic acids in margarine Special. Weigh 9.8 g of margarine, mix it vigorously with 40 g of N32504 and 40 ml

1 M H2504 and distilled with steam, collecting 120 ml of distillate into a receiver containing 10 ml of 1 M NaOH. Half of the distillate is acidified with 1 M H2SO4 to a pH of 2-3, saturated with 30 g of N32S04 and extracted 3 times with 10 ml

® ethyl acetate. The combined extract is dried. 2 g of Na2S04.10ml extract is evaporated to 2 ml. Prepare standard solutions in the same way as in Example 1. On the plate, Silufol UV 254 15 x 15 cm is applied (see chromatogram) in

5 points 1, 2 and 6 in terms of 1.1 and 4 µl of the standard solution, while the amount of BC in the spots is 2.2 and 8 µg, SC, 0.2, 0.2 and 0.8 µg, respectively. At points 3, 4 and 5, 3.3 and 10 µl extract each was added. Points 1 and 3 are treated with 3 μl of a mixture of formic acid and hydrogen peroxide, dried with warm air and placed the plate in a drying cabinet heated to 90 ° C for 15 minutes, cooled and immersed in a chamber with a solvent system: petroleum ether-chloroform-formic acid - diethyl ether 10: 4: 1: 0,8. The plate is examined in UV light with A 254 nm. Chromatograms obtained from points 4 and 5 clearly show spots, corresponding to RF and BK by the value of Rf, while on chromatograms 1 and 3 spots with Rf, BK and CK are absent, but about 3 times smaller than Rf BC. This indicates the presence of a sample of BC and SC. The spots of BK and SC are well separated from impurities and separated. They compare the intensity of the absorption of UV light from the standards and the sample. Based on this assessment, 20 μl of the sample, 10 μl of the standard solution and 20 μl of the blank sample (10 w, ethyl acetate, evaporated to 2 ml) are applied to the Smuufol UV 254 15 x 15 cm plate. The plate is placed in the same solvent system, chromatographed, dried, examined in UV light with 254 nm I, cut out the zones corresponding to the BC and SC in the experimental, blank samples and standards, placed them in test tubes, pour 3 ml of ethyl acetate, the tubes are closed for 3–1 minutes and the optical density of the test sample eluates is measured relatively blank, and standards are measured relative to ethyl acetate on a spectrophotometer in cuvettes with I 10 mm at I 272 nm and 250 nm for BK and SC, respectively.

The calculation carried out according to this formula showed that in the test sample, margarine contains 100 ° C mg / kg benzoic acid and 13 mg / kg sorbinic acid.

PRI me R 3. Determination of benzoic and sorbic acid s block juice.

Apple juice is thoroughly mixed and 10 ml are measured. 5 ml of 1 M NgSSCh and 40 g of anhydrous NaaSCk are added. Intensively stirred and extracted 3 times with 5 ml of ethyl acetate. The combined extract is dried with 1 g of anhydrous NaaSO / j.

On the plate, UV2525 15 x 15 cm is applied (see, chromatogram) at points 1, 2 and b with 1.1 and 4 µl of a standard solution (for preparation of standards, see example 1), while the amount of BK in spots was em 2, 2 and 8 μg, and the UK - 0.2, 0.2 and 0.8 μg, respectively. At points 3, 4 and 5, 6, b and 20 µl of extract are added. Points 1 and 3 are treated with 5 μl of a mixture of formic acid and hydrogen peroxide, dried with warm air and placed in a drying

the cabinet heated to 85 ° C for 20 minutes is allowed to cool and is immersed in a TLC chamber containing a solvent system: petroleum ether-chloroform-formic

acid-diethyl ether 10: 4: 1: 1.6. After chromatography, the plate is examined in UV-light with I 254 nm. On the chromatograms obtained from points 4 and 5, the spots are clearly visible, according to the value corresponding to BSC, Moreover, on the chromatograms obtained from points 1 and 3, the spots with Rf BC and SCs are absent, but Rf appeared to be about three times smaller than Rf BC. This indicates the presence in the sample BC and

Sc. The spots of BC and SC on chromatograms 4 and 5 are separated and well separated from impurities. The absorption intensity of the UV light is compared with the spots of the standards and the sample, and the content of

BK and SC, Taking into account the results of this evaluation, 100 µl of the sample, 50 µl of the standard solution and 100 µl of the blank sample (ethyl acetate) are applied to the plaster of Silufol UV254 15 x 15 cm. Not to overload a thin layer

sorbent, extract, standard solution and blank are applied in several adjacent points. The plate is placed in the same system of solvents, chromatographed, dried, examined in

UV light with I 254 nm, cut out the zone corresponding to the BC and SC in the experimental, blank samples and standards, put them in test tubes, pour 3 ml of ethyl acetate. plugged for three days 1-2 minutes and measured

The optical density of the eluates of the test sample is relatively blank, and the standards are relative etipacetate on a spectrophotometer in cuvettes with I 10 mm at I 272 nm and 250 nm for BK and SC, respectively.

The calculation carried out according to this formula showed that in the sample of the block juice in question, it contains 132 mg / kg of benzoic acid and 877 mg / kg of sorbic acid. Reliable identification and determination of BC is achieved when its content is 2 µg, and SC - 50 ng in the spot. This is at the level of modern methods of analysis and is comparable to the sensitivity of gas and liquid chromatographs.

Claims (1)

The method has been tested on various types of food and beverages (sausage servilat, block marmalade, Special Margarine, tomato and block juice, etc.). Invention Formula The method of quantitative determination of benzoic and sorbic acids in food and beverages, including the isolation from the sample of a food or drink sample containing the benzoic and sorbic acid, deposition on a chromatographic plate with Silufol UV254 sample points and standard solutions, separation of benzoic and sorbic acids by thin layer chromatography in a solvent system containing petroleum ether, chloroform, formic acid in a ratio of 10: 4: 1, identification of spots in UV light, confirmation of the presence of benzoic and sorbic acids in the sample, elution of spots of these acids and spectrophotometric analysis of the eluates at A 272 and 254 nm, respectively, characterized in that. with the aim of improve the reliability of the results and simplify the process by providing a better separation of the benzoic and sorbic acid spots; thin layer chromatography is carried out in a solvent system containing an additional diethyl ether of 0.8-1.6 parts by volume, and confirmation of the presence of benzoic and sorbic acids is carried out by processing points on Start the chromatographic plate with a mixture of formic acid and hydrogen peroxide in a 1: 1 ratio, followed by drying and heating the plate at 80-100 ° C for 15-20 minutes. Table 1 table 2 0.71. 0.65t: 0.55 0.00 f 2 3 Ь 5 6 f tf  99