RU2711410C1 - Method of potentiometric determination of antioxidant capacity of a solution - Google Patents

Abstract

FIELD: measurement technology.SUBSTANCE: invention relates to electrochemical analysis methods, particularly to analysis of solutions for determination of total antioxidant capacity. Invention relates to a method of determining antioxidant capacity of a solution using a potentiometric method, in which a phosphate buffer solution is prepared in advance, to which a system is added which contains an simultaneously oxidized and reduced form of metal in the complex compound K3[Fe(CN)6]/K4[Fe(CN)6], and evaluation of antioxidant capacity is carried out by change in oxidation-reduction potential of solution, measured between working platinum electrode and chloride-silver comparison electrode, recorded before and after introduction into initial solution of analyzed substance. From the total antioxidant capacity of the solution, a reducing and chelating reservoir is recovered, wherein the reducing capacity is pre-determined by potentiometric titration with an oxidized form of the reagent (K3[Fe(CN)6. Chelating reservoir is determined as the difference between the antioxidant capacity and the recovery capacity.EFFECT: obtaining reliable quantitative information on restoring and chelating properties of analyzed objects with antioxidant action, as well as high accuracy and reliability of obtained results.1 cl, 3 ex, 6 dwg

Description

The invention relates to the field of electrochemical methods of analysis, in particular to the analysis of solutions to determine the total antioxidant capacity of the solution.

Methods for the determination of antioxidants using various iron complexes as an oxidizing agent are widespread. They have several advantages: simplicity and informativeness; the availability of a variety of stable iron complex compounds used as an oxidizing agent model. Varying the ligand environment of the composition of the complex compound of iron, it is possible to choose the optimal oxidizing model for oxidizing ability, for the stability of complex compounds at different pH, for solubility. In addition, most iron complexes have a stable hexagonal structure similar to that of the heme in the hemoglobin molecule.

The most important class of antioxidants are polyphenolic compounds. The mechanism of the antioxidant action of polyphenols lies both in their reducing ability and in the fact that polyphenolic compounds can form stable complexes with metal ions of variable valency (stability constants of iron (III) complexes with some polyphenols β = 10 ²⁷ -10 ⁴³ ), and inhibit oxidative processes with the participation of free radicals formed by the Fenton reaction (1):

Fe ²⁺ + H ₂ O ₂ → Fe ³⁺ + OH · + OH ^- (1)

This property is an important chelating component in the total antioxidant effect. Therefore, it is very important to have information about both the restoring and chelating components.

A known method for determining the total antioxidant activity (RU 2282851), which consists in the interaction of the analyte with the reagent Fe (III) -o-phenanthroline, ascorbic acid with the reagent, followed by photometry and calculation of the total antioxidant activity in relation to the standard substance - ascorbic acid.

The disadvantages of this method include the fact that this method does not take into account the chelating capacity of polyphenolic compounds that are part of natural objects with respect to Fe ³⁺ ions, while the Fe (III) -o-phenanthroline complex has a much lower value stability constants (β = 10 ^23.5 ) than many complexes of Fe (III) with polyphenolic compounds. Also, the results obtained by this method are expressed in relative units - equivalents of ascorbic acid.

A known method for the determination of antioxidants in solution [International publication US 6177260В1], based on the use of an Fe (III) -tripyridyltriazine complex as an oxidizing agent, which, when reacted with antioxidants, is reduced to a Fe (II) -tripyridyltriazine colored blue (maximum absorption at 593 nm).

The disadvantage of this method is that the measurements are carried out in an acidic environment; therefore, the method is insensitive to sulfhydryl SH-containing antioxidants, such as glutathione and cysteine, and does not allow us to estimate the total content of antioxidants in the test object. This method also does not take into account the possible formation of complex compounds between polyphenols and iron ions, i.e. the chelating component has not been evaluated. This method, as described above, is spectrophotometric, i.e. does not allow reliable analysis of stained and cloudy samples.

The closest solution is the potentiometric method for determining the oxidant / antioxidant activity of solutions (RU 2235998), which consists in pre-preparing the initial solution into which both the oxidized and reduced forms of the reagent are introduced, and the oxidative / antioxidant activity is assessed by changing the redox potential a solution determined before and after introducing into the initial solution of the analyte.

The disadvantages of this method include the fact that in the case of using the system of free salts Fe ³⁺ / Fe ²⁺ , the possible hydroxo formation is not taken into account, and the chelating effect of polyphenolic compounds is also not taken into account, while when using free salts, the complexation reactions of polyphenols with Fe ions ³⁺ will occur with a higher probability than in previous methods.

Thus, a common problem of existing methods is to obtain an integral parameter about the antioxidant properties of the studied objects and the lack of information about the components of this parameter: chelating and restoring ability of the compounds.

The technical solution of the present invention is to obtain reliable quantitative information about the recovery and chelating capacity of the studied objects with antioxidant action, as well as improving the accuracy and reliability of the results.

The problem is solved by the fact that when determining the total antioxidant capacity of solutions, it is proposed to isolate a reducing capacity and a chelating capacity. When determining the total antioxidant capacity in the solution, there is an excess of a possible complexing agent - the Fe (III) / Fe (II) system in the form of complex compounds, therefore, both the reduction of iron (III) and the complexation between polyphenol compounds and Fe (III) are possible , the probability of which depends on the ratio of the conditional stability constants of the initial iron complexes and polyphenolic iron complexes. Thus, when determining the total antioxidant capacity when interacting with the complex compound Fe (III) in solution, both reducing and chelating capacities are simultaneously determined by reactions (2) and (3).

[Fe ³⁺ L] + AO = n [Fe ²⁺ L] + AO _Ox (2)

During potentiometric titration of the analyzed solution with an iron (III) compound, the interaction occurs under the conditions of a lack of a complexing agent; in this regard, the oxidation-reduction process becomes more likely and, thus, the recovery capacity is determined. The chelating capacity is the difference between the total antioxidant capacity and the recovery capacity.

The essence of the proposed method lies in the fact that the determination of the antioxidant capacity of the solution using the potentiometric method is carried out by changing the redox system of the oxidized / reduced form of iron in the composition of the complex compounds defined before and after the analyte is introduced into the initial solution. The antioxidant capacity in the solution is calculated by the formula:

where AOE is the antioxidant capacity;

With _oh - the concentration of the oxidized form of the reagent;

C _red is the concentration of the reduced form of the reagent;

where E is the initial potential of the system, E ₁ is the potential established in the system after the introduction of the sample, b = 2,3RT / nF,

where R is the universal gas constant, T is the temperature, K, n is the number of electrons involved in the redox reaction; F is the Faraday number.

From the total antioxidant capacity of the solution, a reducing capacity and a chelating capacity are isolated.

The recovery capacity is previously determined by potentiometric titration using the oxidized form of the reagent and calculated by the formula:

where BE is the reducing capacity, C _Ox is the concentration of the oxidized form of the reagent, V _Ox is the volume of the oxidized form of the reagent at the equivalence point, V is the volume of the solution of the analyte,

chelating capacity is estimated as the difference between the antioxidant capacity and the recovery capacity according to the formula:

where XE is a chelating capacity.

Complex reagents Fe (II) and Fe (III) can be used as reagents: K ₃ [Fe (CN) ₆ ], Fe (III) -PHEN, Fe (III) -EDTA, Fe (III) -TPTZ, K ₄ [Fe (CN) ₆ ], Fe (II) -PHEN, Fe (II) -EDTA, Fe (II) -TPTZ.

 The working electrode can be made of platinum, gold, glassy carbon. The reference electrode may be a standard silver chloride electrode.

These differences are significant. Isolation of the chelating and restoring capacity from the total antioxidant capacity allows obtaining reliable data on the antioxidant properties of the studied compounds, the mechanisms of their antioxidant action: restoring and chelating ability.

Currently, from the patent and scientific literature there is no known method for determining the integral antioxidant capacity, which allows to separate the regenerating and chelating capacity in the claimed combination of features.

In FIG. Figure 1 shows the time dependence of the potential upon addition of pyrogallol to the K ₃ [Fe (CN) ₆ ] / K ₄ [Fe (CN) ₆ ] system.

In FIG. Figure 2 shows the integral curve of potentiometric titration of pyrogallol with a solution of K ₃ [Fe (CN) ₆ ].

In FIG. Figure 3 shows the differential curve of potentiometric titration of pyrogallol with a solution of K ₃ [Fe (CN) ₆ ].

In FIG. Figure 4 shows the time dependence of the potential when quercetin is added to the K ₃ [Fe (CN) ₆ ] / K ₄ [Fe (CN) ₆ ] system.

In FIG. Figure 5 shows the integral curve of potentiometric titration of quercetin with a solution of K ₃ [Fe (CN) ₆ ].

In FIG. Figure 6 shows the differential curve of potentiometric titration of quercetin with a solution of K ₃ [Fe (CN) ₆ ].

The method is illustrated by the following examples.

Example 1

In a 10 ml aqueous solution containing 0.01 M K ₃ [Fe (CN) ₆ ] and 0.0001 M K ₄ [Fe (CN) ₆ ] in a phosphate buffer solution, the measuring electrode and the reference electrode are lowered. The steady-state value of the potential (E), measured with a digital voltmeter, is 335 mV. 0.1 ml of a 0.01 M pyrogallol solution is added to the electrochemical cell. The steady-state value of the potential (E ₁ ) is 287 mV.

The measurement results are shown in figure 1. Antioxidant capacity is calculated by the formula:

where AOE is the antioxidant capacity;

With _oh - the concentration of the oxidized form of the reagent;

C _red is the concentration of the reduced form of the reagent;

where E is the initial potential of the system, E ₁ is the potential established in the system after the introduction of the sample, b = 2,3RT / nF,

where R is the universal gas constant, T is the temperature, K, n is the number of electrons involved in the redox reaction; F is the Faraday number.

The calculation shows that the AOE value is 5.17 · 10 ^-4 M-equiv.

In 10 ml of an aqueous solution containing 0.1 mm pyrogallol in a phosphate buffer solution, the measuring electrode and the reference electrode are lowered. Then conduct potentiometric titration of 0.01 M K ₃ [Fe (CN) ₆ ]. The measurement results of the integral curve of potentiometric titration are shown in figure 2. The resulting curve is differentiated by the volume of titrant K ₃ [Fe (CN) ₆ ]. The differential titration curve is shown in figure 3. The volume of added K ₃ [Fe (CN) ₆ ] at the equivalence point, determined from the maximum of the differential titration curve, is 3.8 ml.

Recovery capacity is calculated by the formula:

where BE is the reducing capacity, C _Ox is the concentration of K ₃ [Fe (CN) ₆ ], V _Ox is the volume of K ₃ [Fe (CN) ₆ ] at the equivalence point, V is the volume of the analyte solution.

The calculation shows that the value of BE is 3.80 · 10 ^-4 M-equiv.

Chelating capacity is estimated as the difference between the antioxidant capacity and the recovery capacity according to the formula:

where XE is a chelating capacity.

The calculation shows that the value of XE is 1.37 · 10 ^-4 M-equiv.

Example 2

In a 10 ml aqueous solution containing 0.01 M K ₃ [Fe (CN) ₆ ] and 0.0001 M K ₄ [Fe (CN) ₆ ] in a phosphate buffer solution, the measuring electrode and the reference electrode are lowered. The steady-state value of potential (E), measured with a digital voltmeter, is 338 mV. 0.1 ml of a 0.01 M solution of quercetin are added to the electrochemical cell. The steady state value of the potential (E ₁ ) is 292 mV.

The measurement results are shown in figure 4. Antioxidant capacity is calculated by the formula:

where AOE is the antioxidant capacity;

With _oh - the concentration of the oxidized form of the reagent;

C _red is the concentration of the reduced form of the reagent;

where E is the initial potential of the system, E ₁ is the potential established in the system after the introduction of the sample, b = 2,3RT / nF,

where R is the universal gas constant, T is the temperature, K, n is the number of electrons involved in the redox reaction; F is the Faraday number.

The calculation shows that the AOE value is 4.91 · 10 ^-4 M-equiv.

In 10 ml of an aqueous solution containing 0.1 mm quercetin in a phosphate buffer solution, the measuring electrode and the reference electrode are lowered. Then conduct potentiometric titration of 0.01 M K ₃ [Fe (CN) ₆ ]. The measurement results of the integral curve of potentiometric titration are shown in figure 5. The resulting curve is differentiated by the volume of titrant K ₃ [Fe (CN) ₆ ]. The differential titration curve is shown in Fig.6. The volume of added K ₃ [Fe (CN) ₆ ] at the equivalence point, determined from the maximum of the differential titration curve, is 2 ml.

Recovery capacity is calculated by the formula:

where BE is the reducing capacity, C _Ox is the concentration of K ₃ [Fe (CN) ₆ ], V _Ox is the volume of K ₃ [Fe (CN) ₆ ] at the equivalence point, V is the volume of the analyte solution.

The calculation shows that the value of BE is 2.00 · 10 ^-4 M-equiv.

Chelating capacity is estimated as the difference between the antioxidant capacity and the recovery capacity according to the formula:

where XE is a chelating capacity.

The calculation shows that the value of XE is 2.91 · 10 ^-4 M-equiv.

Example 3

In a 10 ml aqueous solution containing 0.01 M [Fe (III) -o-phenanthroline] and 0.0001M [Fe (II) -o-phenanthroline] in a phosphate buffer solution, the measuring electrode and the reference electrode are lowered. The steady-state value of potential (E), measured with a digital voltmeter, is 446 mV. 0.1 ml of a 0.01 M ascorbic acid solution is added to the electrochemical cell. The steady state value of the potential (E ₁ ) is 417 mV.

Antioxidant capacity is calculated by the formula:

where AOE is the antioxidant capacity;

With _oh - the concentration of the oxidized form of the reagent;

C _red is the concentration of the reduced form of the reagent;

where E is the initial potential of the system, E ₁ is the potential established in the system after the introduction of the sample, b = 2,3RT / nF,

where R is the universal gas constant, T is the temperature, K, n is the number of electrons involved in the redox reaction; F is the Faraday number.

The calculation shows that the value of AOE is 2.04 · 10 ^-4 M-equiv.

In 10 ml of an aqueous solution containing 0.1 mm ascorbic acid in a phosphate buffer solution, the measuring electrode and the reference electrode are lowered. Then conduct potentiometric titration of 0.01 M K ₃ [Fe (CN) ₆ ]. The resulting curve is differentiated by the volume of titrant K ₃ [Fe (CN) ₆ ]. The volume of added K ₃ [Fe (CN) ₆ ] at the equivalence point, determined from the maximum of the differential titration curve, is 2.0 ml.

Recovery capacity is calculated by the formula:

where BE is the reducing capacity, C _Ox is the concentration of K ₃ [Fe (CN) ₆ ], V _Ox is the volume of K ₃ [Fe (CN) ₆ ] at the equivalence point, V is the volume of the analyte solution.

The calculation shows that the value of BE is 2.00 · 10 ^-4 M-equiv.

Chelating capacity is estimated as the difference between the antioxidant capacity and the recovery capacity according to the formula:

where XE is a chelating capacity.

The calculation shows that the value of XE is 0.04 · 10 ^-4 M-equiv. The chelating properties of ascorbic acid are absent.

Claims (14)

A method for determining the antioxidant capacity of a solution using the potentiometric method, which consists in pre-preparing the initial phosphate buffer solution, into which a system containing simultaneously oxidized and reduced metal forms in the complex compound K ₃ [Fe (CN) ₆ ] / K ₄ is introduced [Fe (CN) ₆ ], and the assessment of antioxidant capacity is carried out by changing the redox potential of the solution, measured between the working platinum electrode and the silver-silver chloride reference electrode, striated before and after introducing the analyte into the initial solution, the antioxidant capacity in the solution is calculated by the formula:

, where AOE is the antioxidant capacity; With _oh - the concentration of the oxidized form of the reagent; C _red is the concentration of the reduced form of the reagent;

, where E is the initial potential of the system, E ₁ is the potential established in the system after the introduction of the sample, b = 2,3RT / nF, where R is the universal gas constant, T is the temperature, K, n is the number of electrons involved in the redox reaction; F is the Faraday number, characterized in that from the total antioxidant capacity of the solution, reducing and chelating containers are isolated, while the reducing capacity is previously determined by potentiometric titration using the oxidized form of the reagent (K ₃ [Fe (CN) ₆ ]) and calculated by the formula:

, where BE is the reducing capacity, V _Ox is the volume of the oxidized form of the reagent at the equivalence point, V is the volume of the solution of the analyte, chelating capacity is defined as the difference between the antioxidant capacity and the recovery capacity according to the formula:

, where XE is a chelating capacity.

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