SU1728771A1 - Method of determination of thiols - Google Patents

Abstract

This invention relates to electrochemical methods for the analysis of thiols (compounds of general formula PSH) by electrolysis of a test sample. To simplify and reduce the toxicity of the process, electrolysis is carried out in a 0.1 M potassium phosphate buffer solution, pH 5.5-8.0, using graphite electrodes modified with derivatives of tetracyano-p-quinodimethane, tetratiofulvalene and ferrocene, at a potential of 0, 2-0.1 V Rel, n.e. The anode current is measured, by increasing which the thiol concentration is determined. The method can be used to analyze wastewater and biological fluids. 9 tab.

Description

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The invention relates to electrical methods of analysis, specifically to an improvement in the method for determining the concentration of thiols, compounds containing the SH group, and can be used in medicine, agriculture or other areas of the national economy for analytical purposes,

Thiols - a class of organic compounds containing a carbon group —SH (sulfhydryl or mercapto group). The thiol compounds include two amino acids, L-cysteine and glutathione, which are found in all animal tissues and play an important role in cell biochemistry. Some thiols, such as 2,3-dimercaptopropanol. are used in medicine as antidotes for mouse, mercury and other intoxication poisoning, as well as for cardiac arrhythmias, hepatocerebral dystrophy. Aliphatic amines (2-mercaptoethylamine) are recognized radioprotective agents. Some mercaptans are also used as pesticides, in the manufacture of polymers, and in other areas of the national economy. Therefore, the definition of thiols is important not only in medicine, but also in agriculture, environmental monitoring.

A known galvanostatic method for the determination of thiocholine. The determination is carried out in a 0.08 M Tris-HCl buffer solution, pH 7.4 using a platinum mesh anode and a cathode 1 cm in diameter, previously prepared using a special method (boiling in HMO3 solution, incubation in 3% HaPtCle and 0, 03% lead acetate at a potential of 25 V 5 min. Both electrodes of the platinized plate are obtained. After the first ofX |

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The anode is covered with some material. A constant current of 2 µA is passed through them (the current supply comes from a 9 V battery) and the potential drop at the anode is determined when thiocholine is present in the solution. Thiol concentration is determined from a calibration curve. The nature of the potential difference is not sleep. It can be determined both by the redox transformation of the thiol compound and by the polarization of the anode due to the formation of surface compounds such as platinum sulfides.

The disadvantage of this method is that it uses precious metals (platinum), requires special reagents, some of which are dovita and aggressive (HNOZ. H PtCle, lead acetate). Using this system, it is possible to determine thiol compounds only in pure buffer solutions that lack other electrogenic compounds.

Closest to the proposed one is an amperometric method for the determination of thiols in a borate buffer solution, pH 8.2-8.6 using an amalgamated silver electrode. The amalgamated silver electrode is a silver wire, pressed into or inserted into glass with epoxy resin. To obtain silver amalgam, the end of the electrode is ground to a mirror surface and lowered into metallic mercury for 2 minutes. Amalgamated silver and saturated calomel electrodes are introduced into an electrochemical cell containing 5 ml of a buffer solution of an unknown thiol concentration. Oxygen is removed for 20 minutes and the solution is electrolyzed at a potential of –0.55 vol. Rel. n.e. The concentration is found on the calibration curve.

In the reaction of thiocholine with mercury, mercaptides of the type Hg2 (SR) 2 are formed. where R CH2-CH2-N + (CH3) s, which are reduced on the electrode at a potential of -0.55 V (rel. BC):

Hg2 (SR) 2+ 2H + 2e - - 2RSH + 2Hg

The limiting cathode current is proportional to the concentration of thiol.

The sensitivity of the mercury-silver electrode is 0.011 A / M.

The disadvantage of this method is that it is very toxic, since it involves the use of metallic mercury, a very volatile and poisonous metal, the use of which requires specially prepared and equipped rooms. In addition, the method is non-selective, since the determination is carried out at a potential of 0.55 V rel. AD, in which many impurity compounds in real solutions — oxygen, heavy metal ions (Pb, Ni), quinone and nitro compounds, etc. — are converted. The process takes a long time, since each definition must remove the oxygen that is in all aqueous solutions.

The aim of the invention is to simplify and reduce the toxicity of the thiol determination process.

The essence of the method is explained by the following scheme.

The Moss modifier adsorbed on a graphite electrode reacts with thiols. The reduced form of the MVOS modifier is electrochemically oxidized at the potentials of their redox transformation: Mox + 2RSH-- MBOc + (RS) 2 + 2H +,.

The method is implemented using

graphite electrodes made from rods (5.9 mm in diameter) of spectrally pure graphite. A copper wire is glued to one end of the rod with a length of 3-6 mm with silver epoxy glue, the other end is polished with emery paper (250 µm) and a modifier is adsorbed onto it. The electrodes are pressed into the Teflon case.

0 Example 1. Determination of thiols: L-cysteine, glutathione, mercaptoethylamine and dithiotriethol using a graphite electrode modified with tetracyano-p-quinodimethane (TCHM).

5 The electrode is modified by applying to the surface 40 µl of a solution of TCLC in toluene (6 mg / ml). The solvent is evaporated in air for 2 hours. The electrode is immersed in a thermostated at

0-25 ° С glass cell with 20 ml of 0.1 M potassium phosphate buffer solution, pH 7.0, and using an OH 105 polarograph on a 3-electrode circuit (a modified electrode is used as the working electrode

5, a rotating graphite electrode, a saturated calomel electrode serves as a reference electrode, and a secondary Pt plate serves as the residual current. 50 µl of the thiol solution is injected into the solution. AT

In the course of 30 seconds, a new stationary level of the anode current is established, from which variation the concentration of thiol is determined.

The dependence of the electrode current on the thiol concentration is given in Table 1.

Potential, 15 V Rel. BC, Electrode rotational speed 325 rpm.

As can be seen from table 1, the calibration curves have a linear characteristic up to 0.6-0.7 mM (L-cysteine. Glutathione.

mercaptoethanol, mercaptoethylamine) and 0.25 mm (dithiotrietol). At high concentrations, the calibration curves are bent. The standard deviations in the linear portions of the calibration curves are 0.23 μA for L-cysteine, 0.28 μA for glutathione, 0.27 μA for mercaptoethanol, 0.24 μA for dithiothrysotol, and 0.36 μA for mercaptoethylamine. Sensitivity, i.e. the slope of the straight line is 0.034; 0.014; 0.030; 0.023 and 0.042 A / M for L-cysteine, mercaptoethanol, dithiotriethol, glutathione and mercaptoethylamine, respectively (sensitivity by the known method 0.011 A / M).

Example 2. The electrode is manufactured as in Example 1. The electrode is calibrated with respect to L-cysteine and glutathione at different electrode potentials (Table 2). Concentrations: L-cysteine 0.25 mM, glutathione 0.4 mM. Other conditions as in the example;

As can be seen from Table 2, in the range from 0.3 to-0.1 V, it is practically possible to carry out measurements, but the optimum conditions for carrying out measurements are provided at a potential of 0.15-0.075 V rel. BC, since the ratio of the electrode current to the residual current S / N is greatest at these values of the potential. Below the indicated limit (i.e., 0.2 V), thiol oxidation is not observed, and at a potential greater than 0.2 V, the residual current significantly increases due to electrochemical side reactions at the working electrode.

Example 3. The electrode is manufactured as in Example 1. The electrode is calibrated against L-cysteine glutathione and dithiotrietol at different pH over the entire pH range of the potassium phosphate buffer solution (Table 3). Concentration: L-cysteine, 0, 15 mM, glutathione and dithiotrietol 0.10 mM. Electrode potential 0,15 V rel. n.e. Other conditions as in example 1.

As can be seen from table 3, at a pH of 5.5-8.0, thiol is oxidized, therefore in this range the method can be carried out. However, with the transition from acidic to neutral and weakly alkaline media, the current increases. Consequently, the highest sensitivity of the determinations is provided at pH 7.25-8.0.

Example 4 An electrode is manufactured as in Example 1. The dependence of the electrode current on the electrode rotation speed at a potential of 0.15 V (Table 4) is determined. Other conditions as in example 1.

As can be seen from the table. 4, with an increase in the rotational speed of the electrode, the steady-state current increases slightly. Therefore, a rotational speed of 325 rpm was used in all studies.

PRI me R 5. Determination of the electrode stability in time. The electrode is manufactured as in Example 1. For 2 months, the current is measured in the presence of 0.5 mM L-cysteine. The conditions for determination are as in Example 1. The results of the 0 dependence of the current on the duration of the ° - storage of the electrode are presented in Table 5.

From Table 5 it can be seen that the modified TCLM electrode remains operable for at least 2 months. During this 5 time, the current drops by only 20%.

Example 6. Determination of thiols using graphite electrode modified tetratiofulvalene

(by).--.

0 A graphite electrode with a diameter of 5.9 mM is coated with 40 µl of a solution of TTF in toluene (34 mg / ml). The solvent is evaporated in air for 2 hours, the electrode is immersed in 20 ml of 0.1 M potassium phosphate

5 solution, pH 70.0, and the current is determined. 50 µl of the thiol solution in the same buffer solution is injected into the solution. Within 30 seconds, a new stationary level of electrode current is established.

0 The dependence of the electrode current on the concentration of thiol is given in table 6.

, 1 Rel. n.e. (only in the case of L-cysteine 0.15 V), w 325 rpm, t 25 ° C. As can be seen from table 6, calibration

5 curves have a linear characteristic up to 0.5-0.6 mM (L-cysteine, glutathione, mercaptoethanol and mercaptoethylamine) and up to 0.25 mm (dithiotrietol). At high concentrations, the calibration curves are bent. The standard deviations in the linear portion of the calibration curves are 0.36 μA for L-cysteine, 0.05 μA for mercaptoethylamine, 0.004 μA for glutathione, 0.003 μA for mercaptoethanol, and 0.34 μA

5 for dithiotrietol, and the sensitivity is 0.034; 0.001; 0.003; 0.020 and 0.005 A / M for L-cysteine, glutathione, marcaptoethanol, dithioethol and mercaptoethylamine respectively.

0 EXAMPLE 7. The electrode is manufactured as in Example 6. The electrode is calibrated with respect to L-cysteine and glutathione at different electrode potentials (Table 7). L-cysteine concentration 0.5 mM, and

5 glutathione 0.1 mM. Other conditions as in example 6,

As can be seen from Table 7, both the residual current and the current of the electrode decrease significantly as the potential changes from +0.2 to -0.1 V

rel. n.e. The best ratio S / N in the range of 0.15-0.05 V Rel, n.e.

Example 8. The electrode is made as in Example 6. The electrode is calibrated against L-cysteine, glutathione and dithiotrietol at different pH (Table 8). The concentration of 1-cysteine is 0.4 mM, glutathione 0.9 mM and dithiotrietol 0, 2 mM E 0.1 V relative ke. Other conditions as in Example 6.

As can be seen from table 8, the sensitivity of the electrode significantly increases when moving from acidic to neutral and alkaline.

Example 9. Determination of thiol using a graphite electrode modified with DMFC (53 mg / ml).

The solvent is evaporated in air for 2 hours. The steady state thiol oxidation current is recorded as in Example 1. The dependence of the electrode current on the concentration of mercaptoethylamine in 0.1 M potassium phosphate buffer solution, pH 7.0, at a potential of 0.1 V rel. BC.e., (l) 325 rpm, t 25 ° C is given in table.9.

As is seen from Table 9, the calibration curve has a linear characteristic of 1 mM, the sensitivity in this range is 0.003 A / M, and the standard deviation is 0.03 μA.

The advantage of this method is to simplify the determination of the concentration of thiols that react with the modifier on a graphite electrode to form the reduced form of the mediator, its subsequent electrochemical oxidation and an increase in the anode current. In contrast to the known method, the use of metallic mercury is excluded, which requires a specially prepared and equipped room. Preliminary removal of oxygen before determination, which takes 20 minutes, is also excluded; the electrode used does not respond to oxygen, the way

more specific and expressive.

Claims (1)

Claim method A method for determining thiols, including electrolysis of a test substance in buffer solution and current measurement with subsequent determination of thiol concentration, characterized in that, in order to simplify the process and reduce toxicity, electrolysis is carried out in potassium phosphate the buffer in the range of potentials from 0.3 to -0.1 V Rel. n.e. using a graphite electrode chemically modified by derivatives of tetracyanoquinodimethane, tetrathiofulvalene or ferrocene, and the concentration of thiol is determined by the increase in the anodic current. Table 1 table 2 Table 3 Table 4 Table 5 Table b Table Table 8 Table 9