CZ30183U1 - Working electrode with renewable charge, electrode system and amperometric flow detector comprising thereof - Google Patents

Description

Technical field

The present invention relates to a working electrode with a renewable charge of copper microparticles, preferably spherical, suitable for the determination of organic complexing agents with copper, in particular for electrochemical detection (ED) of amino acids in high performance liquid chromatography (HPLC) and flow injection analysis (FIA) .

BACKGROUND OF THE INVENTION

Due to the importance and frequent occurrence of amino acids, their detection is highly desirable. However, it is complicated by the fact that they usually do not absorb enough in the UV range at normal wavelengths. Thus, in many cases, amino acids need to be pre-derivatized before being detected by UV / VIS spectrophotometry or fluorescence detection (W.T. Kok. U.A.T. Brinkman and R.W. Frei, Journal of Chromatography 1983, 2, 56, 17-26.). Direct detection is only possible using UV detection at 200 nm (I. Molnar and C. Horvath, Journal of Chromatography 1977, 142, 623-640) and using electrochemical methods.

However, direct electrochemical oxidation is only possible with a few aromatic amino acids, such as tyrosine, tryptophan and derivatives thereof. In the case of other amino acids, it is necessary to use a copper electrode for electrochemical detection. Two main electrochemical methods, potentiometry and amperometry, are associated with this working material.

Potentiometry utilizes a complex-forming reaction in the oxide layer on the electrode surface, which leads to a change in the potential of the copper indicating electrode (K. Štulík and V. Pacáková, Electroanalytical measurements in flowing liquids, Ellis Horwood, 1987). The second option is an amperometric arrangement, which is more sensitive and unlike potentiometry does not suffer from non-linearity of calibration dependencies. Amperometry utilizes facilitated dissolution of hydrated oxides on the copper surface in the presence of an analyte complexing with copper ions, leading to an increase in the detector current response. The disadvantage of amperometric detection is the limitation in the field of working conditions. The reaction under investigation takes place only in basic and neutral media (W. T. Kok, B. Hanekamp, P. Bos and R. W. Frei, Analytica Chimica Acta 1982, 142, 31-45) using phosphate and carbonate buffers. Borate buffer, which reduces the rate of the electrode reaction, is not suitable for this purpose. The sensitivity of the assay increases with decreasing concentration of the buffer used (W. T. Kok, G. Groenendijk, U.A. Brinkman and R. W. Frei, Journal of Chromatography 1994, 315, 271-278). Generally, lower flow rates are used which provide sufficient time for the reaction to follow. Another significant drawback is the signal drop during prolonged work with the copper electrode due to the electrochemical dissolution of copper and thus the reduction of its working surface (K. Peckova, V. Mocko, F. Opekar, GM Swain, J. Winter and J. Barek, Chemické Listy 2006 , 100, 124-132).

A variety of copper working electrodes have been developed for electrochemical amino acid measurements. A copper disc electrode was used for FIA and HPLC measurements (W.T. Kok, U.A.T. Brinkman and R.W. Frei, Journal of Chromatography 1983, 2, 56, 17-26). Other detection systems used a copper wire electrode, tubular (K. Stulik, V. Pacakova, M. Weingart and M. Podolak, Journal of Chromatography 1986, 367.3 11-321) and microcylindric copper working electrodes (K. Peckova, V. Mocko, F. Opekar, GM Swain, J. Winter and J. Barek, Chemické Listy 2006, 100, 124-132). Their common disadvantage is the problems associated with the sputtering of the working electrode and the signal drop associated with the dissolution of copper, whose surface is difficult to recover. Therefore, a solution has been sought which allows easy recovery of the working copper electrode surface. Another disadvantage of the previous solutions is the decrease in the detector response over time, which is caused by the dissolution of copper in the electrochemical reaction. Reactivation of the above-mentioned detectors is very difficult and in most cases requires the replacement of the entire working material (disk, wire, tube or microcylinder), which can be time-consuming.

-1 CZ 30183 Ul

The essence of the technical solution

The aforementioned drawbacks of the prior art overcome the present invention, with a refillable working electrode, allowing rapid and simple refilling of the working charge, thereby avoiding passivation electrode or signal loss related to copper dissolution in an electrochemical reaction, significantly enhancing electrode life and quality and related measurement.

The object of the invention is a working electrode with a renewable charge comprising a capillary with an established electrical contact and copper microparticles, preferably spherical, located inside the capillary, and at the end of the capillary containing the copper microparticles a filter fixed at the mouth of the capillary by a screw and endings. The filter serves to retain the copper microparticles inside the capillary and at the same time is permeable to the liquid flowing through the electrode. By changing the size of the copper microparticles, the pore distribution in the working electrode and the hydrodynamic pressure can be modified. The size of the copper microparticles ranges from 1 µm to 1000 µm, preferably from 5 µm to 100 µm. Most preferably, the size of the copper microparticles is from 10 to 20 µm, with smaller particles disproportionately increasing the electrode pressure drop, while using larger particles the degree of conversion decreases.

In a preferred embodiment, the capillary is provided with a flange. The capillary material is preferably Teflon or another thermoformable polymer insoluble in the solvents used. Optionally, a washer may be applied to the capillary to provide a suitable creep of the flattened portion of the capillary, without which the bolt would press only the edges of the flattened portion of the capillary and not the entire surface. The washer includes an opening for passing the capillary through the washer and is preferably made of metal.

Preferably, the filter material is a filter paper that is sufficiently resistant to the organic components of the mobile phases, is readily available, and resistant to the pressure that is generated on the copper microparticles of the working electrode.

In one embodiment, the electrical contact is a platinum, stainless steel or copper wire and / or a tube inserted into a capillary. Preferably, the electrical contact is a platinum tube inserted into the capillary, connected to the platinum wire, since it forms a better contact with the electrode charge of the copper microparticles than the platinum, stainless steel or copper wire itself.

The subject of the present invention is further an electrode system comprising a working electrode according to the present invention, a reference electrode and an auxiliary electrode. In a preferred embodiment, the reference electrode is selected from the group consisting of a calomel, mercurosulfate or argent chloride electrode and an auxiliary electrode selected from the group consisting of a platinum, carbon or stainless steel electrode.

The present invention also provides an amperometric flow detector comprising an electrode system according to the present invention and an overflow cell inside which all three electrodes are located. In a preferred embodiment, all three electrodes are located in a common stand.

The working electrode of the present invention allows for easy filling and replacement of the charge, i.e. the spherical copper microparticles, which can be achieved by reversing the flow or removing the filter. Air bubbles introduced into the carrier solution flow can also be used to accelerate the filling of the working electrode. The detector is preferably filled with a suspension composed of spherical copper microparticles and a carrier liquid, the suspension being introduced into the detector by the flow of carrier liquid through the valve dispensing coil. The surface of the copper particles needs to be activated before filling.

Clarification of drawings

Giant. 1 shows a diagram of a flow-through detector with a renewable charge of spherical copper microparticles according to Example 1.

Giant. 2 shows the hydrodynamic voltammogram - measurement of the response of a detector with a renewable charge of spherical copper microparticles according to Example 1 to the working electrode potential at HPLC-ED alanine (c = 6.10 ⁴ mol.l ^-1 ). Measuring conditions: mobile phase consisting of a phosphate buffer pH 6.8 and methanol (90/10, v / v), volume metering and coils 20 ul mobile phase flow rate of 0.6 mL min ^'first

-2EN 30183 Ul

Giant. 3 shows HPLC-ED records using a refillable copper spherical microparticle detector according to Example 1 taken to measure alanine calibration. Presented are only the HPLC-ED records calibration solutions ranging from 1.10 ^-4 to 1.10 ^{'5 mol.l-1,} namely 1.10 ^-4 mol.l ^-1 (1), 8.10' ⁵ mol.l ^-1 (2) 6.10 ' ⁵ mol.l · ¹ (3), 4.10' ⁵ mol.l ^-1 (4), 2.10 ' ⁵ mol.l' ¹ (5) and 1.10 ' ⁵ mol.l' ¹ (6) (A) and peak height versus concentrations of alanine for the entire range of the measured calibration line, namely from 1.10 'to 1.10 ^{3' 5 mol.l-1} (B). Measurement conditions: mobile phase composed of phosphate buffer pH 6.8 and methanol (90/10, v / v), dosing coil volume 20 μΐ, mobile phase flow rate 0.6 mimin ' ¹ and detection potential +0.2 V .

Examples of technical solution

The invention is further illustrated by the following figures and examples, which do not limit the scope of protection.

Example 1: Electrode system with a working electrode with a renewable charge of spherical copper microparticles

An electrode system with a working electrode with a renewable charge of spherical copper microparticles is shown in FIG. 1.

The electrode system with a working electrode with a spherical copper microparticle filling is based on a Teflon capillary I with an internal diameter of 0.5 mm. The capillary 1 is terminated by a flange (thus it is thermally expanded and flattened and provided with a metal washer 5 which ensures appropriate creep of the flattened part of the capillary). The capillary tube 1 is interrupted in the vicinity of the flange and a 25 mm long platinum tube 8, an outer diameter of 0.6 mm and an inner diameter of 0.5 mm is introduced into it. Between the two parts of the capillary tube is connected to the tube a platinum wire which is separated from the outside by an insulating layer 9 consisting of polystyrene 12 applied by evaporation of its solution in dichloromethane and two layers of shrink tubing 13 and 14. The electrode working material is filled inside the capillary and consists of spherical copper microparticles of about 20 µm in size. The working electrode is filled so that the back pressure of the detector reaches the value of 2 MPa, which is achieved by the filling height of the spherical copper microparticles inside the working capillary about 4 mm.

The electrode system (the working electrode together with the reference electrode 10 (preferably calomel, argent chloride or mercurosulfate) and the auxiliary electrode 11 (preferably platinum, carbon or stainless steel)) is placed in the mobile phase solution 15 in an overflow glass vial (FIG. 1) - all electrodes are placed in a stable stand (not shown in Fig. 1).

When the cartridge 7 is introduced into the working electrode, the working electrode is connected to a pump with two metering valves. The pump generates a flow of the carrier solution (preferably methanol) at a flow rate of 1 mL min ^1st The first valve is used to introduce two 100 μΐ air bubbles into the flow of the carrier solution, thereby reducing the detector filling time. A second valve with an external 500 μΐ loop exchange is filled with a suspension prepared and activated as follows: 0.9 g of spherical copper microparticles are stirred in 3.5 ml of a 0.08 M nitric acid solution and allowed to react for 45 s; at the end of this time, the reaction is stopped by adding 5.5 ml of water. (Thus, 500 μΐ of this suspension is dosed via an external 500 μΐ loop exchange valve).

Example 2: Determination of alanine using an amperometric flow detector with a working electrode with a renewable charge of spherical copper microparticles.

An amperometric flow detector with a working electrode with a renewable charge of spherical copper microparticles according to Example 1 was used to determine alanine by HPLC. The composition of the mobile phase and the electrochemical process of copper activation were taken from the literature (K. Stulík, V. Pacakova, M. Weingart and M. Podolak, Journal of Chromatography 1986, 367, 311-321) and the following mobile composition was used throughout the work Phase: phosphate buffer (0.025 mol.l ^-1 NaH ₂ PO ₄ , pH adjusted to 0.025 mol.l ^-1 NaOH to 6.8) with methanol (90/10, v / v). Under these conditions, other parameters of electrochemical detection, namely flow rate, were optimized

-3GB 30183 UI and Inserted Potential. Alanine solution of 6.10 ^-4 mol.r ^{1 *} prepared by diluting the stock solution (c = 1.10 ³ mol.l ^-1 in 0.1 mol.l ^-1 HCl) with mobile phase was used for all optimization measurements. The detection potential was tested from -0.2 V to +0.4 V against an argent chloride reference electrode using a platinum sheet electrode as an auxiliary. The optimal potential was +0,2 V, where the maximum detector response. The optimum flow rate was chosen from 0.6 ml.min ^-1 , from the test interval 0.3 to 1.0 ml.min ^-1 . The values of peak areas and heights were similar in the whole measured interval. The flow rate of 0.6 ml.min ^-1 was therefore chosen especially with regard to the duration of the individual analysis and the working pressure in the detector.

Under the optimum conditions obtained, the calibration dependence was measured and the dynamic linear range, the limit of detection and the limit of determination were calculated, which were calculated as three and ten times the standard deviation of the determination of the lowest monitored concentration. The results obtained are summarized in Table 1.

In FIG. 2 depicts the response of a working electrode with a renewable charge of spherical copper microparticles to the detection potential at HPLC-ED alanine (c = 6.10 ^-4 mol.l ^-1 ).

Giant. 3 shows HPLC-ED records using a spherical copper microparticle refillable electrode taken for alanine calibration.

Table 1. Selected parameters for determination of alanine by HPLC-ED with amperometric flow detector with working electrode with renewable charge of spherical copper microparticles

Detection limit (molT ^-1 ) 4.05 TO ^-4 Determination limit (mol Γ ') 1,35-10 ^-5 The correlation coefficient 0.998

Example 3: Surface Activation Procedure of Copper Microparticles

0.9 g of spherical copper microparticles are stirred in 3.5 ml of a 0.08 M nitric acid solution and allowed to react for 45 s; at the end of this time, the reaction is stopped by adding 5.5 ml of water. The activated mixture is stable for at least 24 hours. Thus, the resulting suspension suitable for filling the detector has a composition of 10 mg of copper per ml of dilute nitric acid aqueous solution. The suspension thus prepared can be advantageously filled into the detector by means of a dispensing coil of 500 μΐ, from which it is eluted into the working electrode with a suitable carrier solution. Methanol at a flow rate of 1 ml.min ^-1 may advantageously be used as the carrier solution for this filling. Filling can be further accelerated by introducing two 100 μΐ air bubbles into the methanol flow. This can be achieved by upstream of the second metering valve into the system just before the valve serving to fill the spherical copper microparticle suspension.

Industrial applicability

Electrochemical detection using an amperometric flow detector filled with spherical copper microparticles can be used to determine organic compounds that are capable of forming complex compounds with copper, especially amino acids, by flow methods, particularly HPLC and FIA.

PROTECTION REQUIREMENTS

Claims (7)

Renewable charge working electrode, characterized in that it comprises a capillary (1) with an established electrical contact (2) and copper (7), preferably spherical, microparticles (7) located within the capillary (1) and at the end of the capillary (1) 1), comprising copper microparticles (7), a filter (6) is provided, fixed at the mouth of the capillary (1) by means of a screw (3) and an end piece (4). -4EN 30183 Ul Working electrode according to claim 1, characterized in that the capillary (1) is provided with a flange. Working electrode according to claim 1 or 2, characterized in that the material of the filter (6) is filter paper. A working electrode according to any one of the preceding claims, characterized in that the electrical contact (2) is a platinum, stainless steel or copper wire and / or a tube inserted into the capillary. An electrode system, characterized in that it comprises a working electrode according to any one of the preceding claims, a reference electrode (10) and an auxiliary electrode (11). ío Electrode system according to claim 5, characterized in that the reference electrode (10) is selected from the group consisting of calomel, mercurosulfate and argent chloride electrodes. Electrode system according to claim 5 or 6, characterized in that the auxiliary electrode (11) is selected from the group consisting of a platinum, stainless and carbon electrode. An amperometric flow detector comprising an electrode flow detector A system according to any one of claims 5 to 7 and an overflow cell, within which all three electrodes are located.