DE2924117A1 - ELECTRODE SYSTEM WITH A REFERENCE ELECTRODE WITHOUT LIQUID TRANSITION FOR VOLTAMETER MEASUREMENTS - Google Patents

Description

Istituto Sieroterapicö e vaccinogeno Toscano "SCLAVO" S.p.A.

Siena / Italy

Electrode system with a reference electrode without smooth transition for voltmeter measurements

The invention relates to a new electrode system which enables polarographic or voltmeter measurements to be carried out in a particularly advantageous manner.

The voltameter system at hand consists of a working electrode, a reference electrode and an auxiliary electrode, immersed in a solution with constant ion concentration,

with respect to which the reference electrode is reversible. The reference electrode is an electrode of the second type or is of the metal / metal oxide type.

It is well known that the recording of current / span diagrams, as obtained under suitable conditions, forms the basis of an important analytical method called polarographic or voltameter analysis. This method not only allows simultaneous determination both qualitatively as well as quantitatively numerous cations and anions and also a large number of organic substances that are electrochemically
can be reduced and oxidized, but is also able to solve various problems of chemical nature, such as those of a constitutional determination of certain substances,

the investigation of reaction mechanisms, the identification of

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Equilibrium constants, the determination of stability constants of complexes and numerous others.

The electrode systems used in polarographic measurements can generally consist of 2 or 3 electrodes.

The first pail is the electromotive force between the working electrode and the reference electrode are applied and the current flowing between the two electrodes is measured.

The second pail is made using a potentiostatic System applied an electromotive force zv / ischen an auxiliary electrode and the working electrode in such a way that the desired electromotive force between the working electrode and the reference half-cell is present.

The current flowing through the working electrode is dependent the preselected electromotive force is recorded.

As a rule, a three-electrode system consists of:

1. the working electrode (a drop of mercury, Pt, Au, graphite and others)

2. the auxiliary electrode (Pt)

3. the reference half-cell (Hg / Hg ₂ Cl ₂ / KCl)

(Hg / Hg ₂ SO ₄ / K ₂ SO ₄ ) (Ag / AgCl / KCl)

The most serious of the use of reference half-cells Problems arising with an inside solution and a fluid compound are:

The physical dimensions cannot be reduced beyond certain limits , so that the use in kl el ::: a system becomes difficult;

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the liquid connection between the solution to be examined and the solution inside the half-cell causes the so-called "liquid connection potential", which is laboriously reduced this potential also varies depending on the nature of the solution to be investigated; the sample of the solution to be examined is contaminated by the solution inside the half-cell and vice versa; the reference half-cell requires comparatively frequent maintenance measures.

According to the invention it has been found that it is possible to use the conventional Replace the reference half-cell with a fixed electrode, for example one of the second type, its potential by a constant ion concentration (with respect to which the electrode in question is rerersible), which is introduced into the investigating solution is introduced, is stabilized.

Usable electrodes can be the following: Iridium / Iridiumoxid, Palladiuta / Palladiuraoxid, Rhodium / Bhodiuraoxid (with a constant H 2 concentration in the solution to be examined); a fluoride electrode (with a constant P ~ concentration in the solution to be examined); Ag / AgCl, Ag / AgJ and Ag / AgBr (with a constant concentration of Cl "" -, J ~ "or Br ~~ ions in the solution to be examined); Ag / AgpS (with a concentrate! O:
seeking solution).

a concentration of either Ag ⁺ or S ~ in the

Using the electrode system according to the invention, which can contain, for example, a working electrode and an auxiliary electrode both made of platinum and an Ir / Ir0 ₀ reference electrode (as shown in Pig. I ), it is possible to initiate an electrochemical reaction at the Arboitnel electrode when working in a buffer solution see below about: ;;; 2'α ·? Ώ, which rr.ivor has been charged to a predetermined voltage with respect to the reference electrode.

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The electrode system according to the invention has the advantage that it is not affected by any of the deficiencies listed above for systems using reference hair cells with a solution inside will. A particular advantage is based on the fact that the 3 individual electrodes quickly become a single unit can be arranged even if they have a reduced size and measurements of volumes in the microscopic hate rod can be adapted to the solution to be investigated.

Figure 1 of the accompanying drawings shows only by way of example a possible embodiment of the present electrode system.

Reference is now made first to FIG. In this means the number 1 is the Ir / IrOg electrode, 2 is the platinum working electrode, 3 the auxiliary platinum electrode, 4 a probe (if applicable) for temperature control, 5 is the body of the electrode system and 6 is finally the arrangement of the connection parts for connecting the measuring instrument. The electrode system, as now described, can be used to carry out several Analyzes are used. E.g. it can be used for the following determinations be used:

1. Determination of hydrogen peroxide, e.g. in connection with the development of the following enzymatic reactions:

1.1, O ₂ + aD-glucose

1.2. O _{2 +} alcohol ^aldehyde 7 ^d + ^H ₂ ° 2

1.3. O ₂ + uric acid P ^ricase > allantoin + CO ₂ + H ₂ O ₂

1.4. O ₀ + D-galactose - r D-galactohexodialdo-2 oxidase - _se + H ₉ O ₂

1.5. O ₀ + cholesterol 4-cholestan-3-one + iU

1.6. O ₀ + H ₀ O + L-amino3 acid 2-sefco acid +

^ά - uxyaase NH., + H "0 _o

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2. Determination of ΙΓΑΌ (Ρ) Η ^ == ^ ΐίΑΙ) (Ρ) ⁺ , for example in connection with the development of the following enzymatic reactions:

2.1. Pyruvab + ITADH L-lactate + ITAD ⁺

Tefehydrogenase

2.2. L-glutamate + H _p O + HAD (P) ⁺ 2-ketoglutarate

Dehydrogenase + 1TH, + IIAD (P) H

2.5. Alcohol + HAD (P) ⁺ aldehyde + ITADH

Dehydrogenase

2.4. Glycerin + ITAD ⁺ ■ -> Dihydroxy ac et on-f ITADH

tfehydr ο genäse

2.5. L-malate + irAD ⁺ oxaloacetate + UAIH

Dehydrogenase

2.6. β-D-glucose + ilAD (P) ⁺ D-gluconolactone +

tfehydrogenase IiAD (P) H and others

3. Determination of biological and non-biological substances Substances that are already present in the liquids to be examined in contrast to those that are formed during reactions like the ones mentioned before, such as:

3.1. uric acid

3.2. Ascorbic acid

4. Determination of all substances that are usually detected using conventional electrode systems.

Further process details will become apparent from a study of the following examples which further illustrate the invention.

example 1

Determination of glucose according to the above under 1.1. specified Reaction scheme.

9 0 9 8 5 1/0 8 7 5

y ■ / ■ λ 117

Materials;

1. Enzymatic reagent containing 75 mM phosphate buffer, pH 7 and glucose oaydase 50 U / ml

2. Commercially available control sera

3. Glucose standard solutions (5Ο-1ΟΟ-15Ο-2ΟΟ-3ΟΟ-5ΟΟ mg / dl) in saturated benzoic acid solution

4. Eigur 2 measuring cell connected to the special polarographic instruments.

In Figure 2, the reference number 1 indicates the way of introducing the enzymatic reagent via an automatic metering device on, 2 are the electrodes connected to the polarograph, 3 is the sample feed pipette, 4 is a stirrer, 5 is the one with lowering connected to a suction system, 6 are the connections between the sensors and the polarograph.

Methods;

Van brings 2 ml of enzymatic reagent into the measuring ropes and adds 0.02 ml of serum or a standard see below. The Polarograph is not connected to a recording device for recording the course of the reaction on a diagram. One obtains peaks like never shown in Figure 3. The height of each peak is proportional to the concentration of glucose in the sample (Figure 4).

'/ -MT check the accuracy were examined eight commercial sera and the results are given in Table I below.

Comparison with the enzymatic-colorimetric method (GOD-POD) resulted in the following data:

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Number of samples Equation of the regression line Correlation coefficient mean of χ mean of y maximum of χ Maximum of y Minimum of χ Minimum of y Area of χ Area of y

y = 0.94 x + 7.8 r = 0.985 128.5 mg / dl 128.0 mg / dl 325.0 mg / dl 341.0 mg / dl 57.0 mg / dl

57.0 mg / dl 268.0 mg / dl 284.0 mg / dl

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Iabelle I

• items ^A Art. · ■ 701 Oil. 4th • geraessei Specified
glucose i 76.4-86.4 mg / dl) Found
glucose It HYLAND normal ^ Article 763 82.O mg / dl '( ti ae values 75.0-89.0 It) 35.O mg / dl, - It CLINICONTROL items IRO13N. IO4.O Il j Il Il 93.0-115.0 11 p • 33.7 ti PRECILIP items IROI3B, 86.6 Il j Il Il 80.0-92.2 11 · \ 17.5 Il ORTHO I ' items IRO13P- 126.0 It I ti Il 115.0-137.0 Il) 82.5 ti ORTHO II. items PÖ2- 3O5.O Il j It It 280.0-330.0 It) 117.5 ' H ORTHO III HYLAND Abnormal Art. 702 200.0 '· «.'. ( It Il l88.Or212.O It) 285.0 It CLINICONTROL I99.O Il j Il Il I82.O-2I6.O It) 213.O It ( Il 200.0

Example 2

Determination of NADH in the presence of ITAD ⁺

A polarographic system was used with the following process parameters used:

Voltage: + 800 mV

Full scale value: 500 nA (nanoampere)

Mechanical stirring: at approx. 500 TJpM

Reaction volume: 10 ml phosphate buffer 0.05 M, pH 7.4

^1. To

Increasing amounts of 10 mcl to 100 mcl (millicentiliter J) sample of 0.1 mg / ml NADH were given and a continuous recording of the current intensity in relation to the oxidation reaction of NADH to NAD ⁺ was carried out.

The instrument's readings were proportional to the increase in NADH concentration in the reaction cell with a 70 nA signal for 0.14 mmol / ml NADH.

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Claims (2)

Dr. F. Zumstein Sr. - Dr E. 'Λ ss-n ^ in - D »-. F, ^ oenigsberger Dipl.-Phys. R. Holzbauer - Dipi.-Jng. F. Klingseisen - Dr. F. Zumstein jun. PATENTANWÄLTE aOOO Munich 2 Brauhausstrasse 4 Telephone collection no. 225341 · Telegrams Zumpat ■ Telex 529979 Case 1164 Λ Patent claims 1.'electrode system consisting of a working electrode, a reference electrode and an auxiliary electrode immersed in a solution which contains a constant ion concentration, "with respect to which the reference electrode is reversible, the reference electrode being an electrode of the second type in a solid state . 2. Electrode system according to claim 1, characterized in that the three electrodes all together in a fixed beam component are built in. 909-85 1/087 B.