EP2791662A1

EP2791662A1 - Reference electrode

Info

Publication number: EP2791662A1
Application number: EP11808184.3A
Authority: EP
Inventors: Rolf Thrier
Original assignee: Mettler Toledo AG
Current assignee: Mettler Toledo Schweiz GmbH
Priority date: 2011-12-15
Filing date: 2011-12-15
Publication date: 2014-10-22
Also published as: CN103975235A; US9134266B2; WO2013087106A1; US20140034515A1

Abstract

The invention relates to a reference electrode having an in-situ modified porous diaphragm and to a method for in-situ modifying the porous diaphragm, wherein the reference electrode comprises at least one housing (1, 201, 301), a first discharge element (4, 204, 304), a modified electrolyte, which is free-flowing and is disposed in the housing (1, 201, 301), and a porous diaphragm (3, 203, 303), said porous diaphragm providing a fluidic connection between the modified electrolytes and a measurement medium (9, 209, 309), and through which the modified electrolyte is discharged during operation, wherein the modified electrolyte comprises a first component and a surface-modified component that in-situ modifies the surface of the porous diaphragm (3, 203, 303) as the modified electrolyte passes through.

Description

Ref e re e ctrod e

The invention relates to a reference electrode for an electrochemical measuring chain with a porous diaphragm whose surface is modified in-situ.

Reference electrodes are used in numerous electrochemical sensors and / or measuring chains, for example in potentiometric or amperometric sensors. These electrochemical sensors are used in the laboratory as well as in various industries, such as the chemical industry, the food industry, biotechnology or the pharmaceutical industry.

It is essential for such reference electrodes that they deliver as constant a reference potential as possible.

Known reference electrodes include, for example, a housing in the interior of which a reference electrolyte is located, via an overpass, too

Liquid connection or called "liquid junction", in contact with a

Measuring medium is.

Furthermore, reference electrodes are known, which in addition to the reference electrolyte also comprise a bridge electrolyte, wherein the bridge electrolyte via a transfer, also called liquid connection or "liquid junction", is in contact with a measuring medium and via a further transfer with the

Reference electrolyte is in contact. Such dual-chamber systems are used in particular when the electrolyte must not come into direct contact with the measuring medium.

Particularly in the bio, pharmaceutical or food sector, high demands are placed on the safety, cleaning and sterilizability of the reference electrode or the sensor and the materials contained therein. The substances and materials coming into contact with the measuring medium should be chemically inert towards the measuring medium and preferably harmless, in particular non-toxic. The reference electrodes should be easy to clean and sterilize. The known cleaning methods include, for example, CIP and / or SIP cycles, the be carried out under high temperatures with highly concentrated alkalis or acids. Furthermore, the so-called biofouling, ie deposits and deposits of foreign substances or interfering substances should be prevented in particular on the diaphragm surface. The transfer, for example, be an open transfer or be designed as a porous diaphragm. The transfer should, on the one hand, have the lowest possible electrical resistance and, on the other hand, stop the mixing of the reference or bridge electrolyte and of the medium to be measured

trying to achieve different measures. For example, US Pat. No. 7,387,716 B2 or EP 1 560 019 B1 disclose

Reference electrodes with open transfer in combination with a solid or solidified reference electrolyte, for example a polymer electrolyte. In this way, an uncontrolled outflow of the reference electrolyte can be prevented or at least greatly reduced by the open transfer into the measuring medium. However, the polymer electrolytes used often consist of organic

Compounds which are not always safe or may even be toxic.

Also known is the use of porous diaphragms as transfer,

preferably in combination with a flowable electrolyte. The porous one

Diaphragm can also prevent or at least greatly reduce the uncontrolled outflow of the reference or bridge electrolyte by the transfer into the medium. Porous diaphragms can be used, for example, in

so-called printed reference electrodes are used, as disclosed inter alia in DE 3 702 501 A1. These are designed such that the reference electrolyte exits continuously through the diaphragm from the pressurized reference electrode due to an increased internal pressure. So the

Reference electrolyte is not consumed too fast, the pores of the diaphragm should be as small as possible. By the pressurization, both a possible penetration of the measuring medium and an addition or clogging of the pores of the diaphragm by the exiting reference electrolyte can be prevented or at least reduced. However, reference electrodes with porous diaphragms also show disadvantages when used in biomass or protein-containing measuring media. An essential one

Difficulty in the use of reference electrodes in protein-containing measuring media is the so-called biofouling. Most naturally occurring proteins have a negative charge at neutral pH and thus have an affinity for adsorption to oxidic surfaces. Frequently, diaphragms consist of oxidic substances, such as, for example, oxidic ceramics, so that they can be contaminated and / or clogged, in particular if small-pore diaphragms are used, in particular in protein-containing measuring media. Diaphragm contamination by protein-containing measuring media leads to transfer

Failure potentials and resulting measurement errors, whereby the tendency to contamination increases with decreasing pore size. It can even lead to erroneous measurement signals or to a complete failure of the reference electrode. Even the increased internal pressure of a printed reference electrode and the associated outlet of the reference or bridge electrolyte are not sufficient for efficient cleaning of the diaphragm. In addition, a contamination of the

Measuring medium through the emerging reference or bridge electrolyte or by deposits on the diaphragm possible. Advantageously, however, the diaphragm should also be free from contamination during use in protein-containing measuring media or in biomass during operation.

The known solutions for avoiding or reducing biofouling and in particular the protein contamination of sensor surfaces and diaphragms have disadvantages. When pressurized reference electrodes, the purging of the porous diaphragm is possible only with a relatively high discharge speed, resulting in a high consumption of reference or bridge electrolyte and thus a

close-meshed electrode maintenance for refilling the reference or

Bridges electrolyte pulls. With thickened and thus less flowable reference or bridge electrolytes cleaning of the diaphragm by flushing can not be achieved, also many of the thickening agents used are organic substances that can not be classified as harmless and can contaminate the medium, for example. The reference or bridge electrolyte can of course also be used with naturally occurring and substantially acceptable polymers, such as agar-agar or cellulose, but these substances do not withstand the specified

Cleaning procedures.

The object of the invention is therefore the development of an improved

Reference electrode, which provides reliable and reproducible results even when measurements in biomasses and protein-containing measuring media and is also resistant to the known cleaning methods and process conditions.

This problem is solved by a reference electrode for electrochemical

Sensors comprising a housing, a discharge element, a flowable modifying electrolyte disposed in the housing and a porous diaphragm. The porous diaphragm constitutes a fluid connection or a transfer between the modifying electrolyte and a measuring medium, the modifying electrolyte exiting through the diaphragm during operation. The modifying electrolyte comprises a first component and a surface-modifying component which surrounds the surface of the diaphragm as it passes through

modifying electrolytes modified in situ.

The in-situ modification of the diaphragm surface is advantageous over a factory modification, since this can be carried out in the operation of the reference electrode, ie in-situ, and an aging and / or cleavage of a before in

Commissioning applied surface modifying component of

Diaphragm, for example, during the implementation of CIP and / or SIP processes or when using aggressive measuring media, can be largely prevented.

The reference electrode diaphragm is made of a porous material having continuous pores in the interior of the diaphragm. The surface of the

Diaphragms referred to herein thus not only designates the surface facing the measuring medium but also the surface provided by the pores. The first component of the modifying electrolyte may be an electrolytically conductive and / or potential-determining substance, as frequently used in or as a reference or reference electrolyte.

The first component is electrolytically conductive, wherein preferably equitransferent salts are used, their anions and cations in aqueous solution in

Have substantially the same diffusion rate. The use

Equitransferenter salts is advantageous because so disturbing diffusion potentials can be reduced at the transfer.

In reference electrodes of the second type, the first component may comprise a chloride compound such as KCl, NaCl, MgCl ₂ or CsCl ₂ .

In dual-chamber reference systems with a bridge electrolyte as

modifying electrolyte, the bridge electrolyte may include, for example, Na ₂ S0 ₄ as a first component. Of course, other known as reference or bridge electrolytes substances as the first component of

modifying electrolytes are used. In pH reference electrodes, the first component comprises a pH buffer system and in redox reference systems a redox buffer. The first component of the modifying electrolyte becomes

preferably used in the form of aqueous solutions, in particular an aqueous potassium chloride solution is used, preferably in a concentration of 3 mol / L.

The in-situ modification of the porous diaphragm serves to prevent or at least greatly reduce biofouling on the surface of the porous diaphragm, which has already been attempted by various means. Porous diaphragms can be made, for example, with an inert polymer,

For example, PTFE (polytetrafluoroethylene), or a hydrophilic cross-linked hydrogel are coated. Unfortunately, these layers are difficult to apply, partially dissolve easily and are also relatively thick, reducing the pores the porous diaphragms become too narrow, which may result in undesirable, elevated diaphragm resistances.

Another possibility is the occupancy of the diaphragm surface with covalently bonded substances, which has the disadvantage that the resulting covalent bonds are often not resistant to hydrolysis compared to the acids and bases used for example for cleaning and the applied layers in some cases already after a single Replace CIP treatment. Silanes were used as substances, for example. In particular, the Pegylierung by silanes with PEG residue (polyethylene glycol), which relatively completely prevent the protein addition, but also subject to the disadvantages shown.

To overcome these disadvantages of the prior art, the modifying electrolyte comprises a surface-modifying compound in addition to the first component

Component which, by virtue of its characteristics, is capable of in situ modifying the surface of the diaphragm so as to target proteins and / or

Repulsive amino acids acts. The modification takes place in-situ, so that it is also possible to use substances as surface-modifying components which bind reversibly to the surface and thus change the surface charge. Preference is given to surface-modifying substances which also bind quickly to the surface. The surface-modifying component is added to the modifying electrolyte exiting the diaphragm during operation so that the diaphragm surface, during operation of the diaphragm

Reference electrode, a uniform occupancy with the surface-modifying component, even when using a reversibly adsorbing surface-modifying component or when using a substance as a surface-modifying component, which are replaced, for example by the usual cleaning method of the diaphragm surface.

In particular, the surface-modifying component used should be non-toxic. This is particularly important when the inventive

Reference electrode for measurements in biomass or protein-containing solutions to be used. Advantageously, the reference electrode is configured such that in use the modifying electrolyte continuously exits through the porous diaphragm so that the surface-modifying component can continuously modify the surface of the diaphragm. This refinement of the reference electrode is particularly advantageous, since the continuous passage of the modifying electrolyte through the porous electrolyte

Diaphragm on the one hand mechanically cleaned by the modifying electrolyte flushes out foreign substances from the porous diaphragm in the passage, and on the other hand, in this way, the surface modification can be continuously renewed or supplemented. This is particularly advantageous in the use of a surface-modifying component which reversibly binds or physisorbs to the diaphragm surface. For example, reversible bonding may be via hydrogen bonds, electrostatic interactions, or Van der Waals forces. In order to ensure and preferably also to control the continuous passage of the modifying electrolyte through the porous diaphragm, the

Reference electrode be pressurized. Due to diffusion and capillary forces, even with a non-pressurized reference electrode, the diaphragm surface would be slowly coated with the surface-modifying component in the modifying electrolyte or it would be on the surface thereof

adsorb. As a result of the pressurization, the modifying electrolyte flows through the diaphragm at a constant exit velocity, so that new surface-modifying components are continually introduced through the pores of the diaphragm

Diaphragm is passed. As a result, an in-situ and in particular a

continuous modification of the diaphragm surface allows. In addition, a relatively fast in-situ modification can be achieved.

In one embodiment of the reference electrode, the modifying electrolyte may be a reference electrolyte into which the diverting element is immersed.

In a further embodiment of the reference electrode, the modifying electrolyte may be a bridge electrolyte, which via the diaphragm with the Measuring medium is in contact. In this embodiment, the reference electrode also has a reference electrolyte and a reference housing with a further diaphragm, wherein the diverting element dips into the reference electrolyte and the reference electrolyte is in contact with the bridge electrolyte via the further diaphragm. Such reference electrodes can, for example, a

Dual chamber system comprising an inner and an outer electrolyte, wherein the outer electrolyte, the bridge electrolyte, with the measuring medium in contact via a diaphragm and comprises the surface modifying component. A further embodiment is the embodiment of a reference electrode according to the invention as a pH electrode, which is a pH buffer system as the first

Component of the modifying electrolyte comprises. Such systems are also referred to as the differential pH measuring chain. The pH buffer system can be the first

Component comprise a potential determining substance, such as an acetate or citrate buffer. As the surface-modifying component, one of the already mentioned substances can be used.

Substances which adsorb or deposit on the diaphragm surface and subsequently inhibit the protein absorption or the addition of other or further foreign substances can be used as the surface-modifying component. Preferably, the substance used as surface-modifying component is non-toxic and chemically inert to the measured medium. The subsequent adsorption of foreign substances or interfering substances, such as proteins, can be inhibited either by electrostatically acting or by sterically demanding substances. Electrostatically active substances change the surface charge of the

Diaphragm, which inhibits the adsorption of foreign substances on the diaphragm surface and thus prevents or at least reduces biofouling. Adsorption on the diaphragm surface is enhanced or accelerated when attractive surface forces prevail for the adsorbent, for example proteins. The diaphragm surface modified by an electrostatic substance has a charge or forces repulsive for adsorption, so that disturbing protein adsorption on the diaphragm surface is electrostatically inhibited. It should be noted that the charge of the diaphragm, as well as the adsorbent is often a pH-dependent size.

In a further embodiment, a sterically demanding substance can be used as a surface-modifying component which can sterically hinder or even prevent the addition of substances contained in the measurement medium, in particular of proteins. As sterically demanding substances are referred to, which can significantly hinder the adsorption of other substances on the diaphragm surface by their space filling. The steric hindrance can also affect the reaction kinetics and, for example, slow down competing adsorptions, since the adsorption of further substances by the already adsorbed bulky substance can be prevented. An example of a sterically demanding substance is polyethylene glycol (PEG).

In further embodiments, the surface-modifying component may be an organic substance comprising at least one hydroxy substituent and at least one carbonyl substituent. These substances can u. a. bind to the diaphragm surface via hydrogen bonds and thus inhibit the adsorption of other substances, such as proteins.

Examples of such organic substances are organic acids, such as

Lactic acid, citric acid, malic acid, tartaric acid, ascorbic acid, their salts or mixtures thereof. These compounds all have at least one hydroxyl and one carbonyl substituent and are also distinguished by being non-toxic. The exemplified carboxylic diacids are negatively charged and, due to their intrinsic charge, adsorb particularly effectively on positively charged diaphragm surfaces.

Malic acid, ascorbic acid or other reducing substances are preferably used in reference electrodes with silver chloride-free modifying electrolyte, since these can cause an undesirable reduction of silver chloride to silver, especially at elevated temperatures. Surprisingly, it has been found that in particular citric acid or its salts due to their reduction potential reference electrodes with

Silver chloride-containing modifying electrolyte, even at elevated temperatures substantially not change. Therefore, in particular citric acid, as well as their salts, are suitable for use as a surface-modifying component in Ag / AgCl reference electrodes.

Another embodiment of the reference electrode may include polylysine-polyethylene glycol (PLy-PEG) as a surface-modifying component. This substance can adsorb by means of its polyamino acid part on an oxidic surface, after which the surface facing away from the PEG part can prevent the adsorption of other interfering foreign substances or interfering substances. As foreign substances, for example, proteins and other molecules from a biomass are referred to, which adsorb on a blank diaphragm surface or occupy it, thereby clogging the diaphragm and thus make it useless. Preferably, the surface-modifying component is added to the modifying electrolyte in a low concentration of less than 0.1 weight percent (wt.%), And more preferably from about 0.01 to 0.1 wt.%. These

Concentration is sufficient to ensure a modification of the diaphragm surface, and at the same time is so low that the occurrence of disturbing diffusion potentials by the added surface-modifying component in the

Substantially prevented or at least greatly reduced.

For example, the porous diaphragm may comprise a ceramic material or a metallic structure having a large surface area.

Zirconia-containing ceramics are preferably used, since they are particularly stable to alkalis, ie in particular also stable with respect to the known ones

Cleaning methods are, and therefore are particularly suitable for use as a porous diaphragm in a reference electrode. Furthermore, aluminum-silicate ceramics can be used as porous diaphragms, but these are much less resistant to the usual cleaning methods.

An example of a metallic structure is a bundle, capillaries or balls of metal wires, which consist for example of a noble metal and in particular of passivated platinum.

Both the listed ceramic materials and metallic structures are characterized by a high resistance to the common

Process conditions and cleaning process off. Another aspect of the invention relates to a method for the in-situ modification of a porous diaphragm, which is arranged as a transfer or fluid connection in a reference electrode according to the invention. This method includes, among other things, adding a surface-modifying component to a modifying electrolyte disposed in the reference electrode, as well as ensuring the exit of the modifying electrolyte through the porous diaphragm. In this way, the surface of the diaphragm of a

Reference electrode in operation - ie in situ - be modified and a clogging and / or contamination of the diaphragm by others, for example in

Measuring medium existing substances are effectively prevented. In an advantageous embodiment, the reference electrode is pressurized to a continuous passage of the modifying electrolyte through the

To ensure diaphragm.

Various exemplary embodiments of a reference electrode according to the invention and the method for the in-situ modification of a porous diaphragm of a reference electrode according to the invention are explained below with reference to the figures, wherein the same features are provided with the same reference numerals. The exemplary embodiments are described in particular with reference to a pH measuring chain with a reference electrode according to the invention which comprises a reference electrolyte. The term reference electrolyte will therefore be partially in the following used synonymously with the term modifying electrolyte. A

Of course, reference electrode according to the invention can also be used with other electrochemical measuring chains or sensors. The figures show:

Fig. 1 is a schematic representation of a combined pH electrode with a

Reference electrode in longitudinal section;

Fig. 2 is a schematic representation of a combined pH electrode with a

Reference electrode in longitudinal section, wherein the reference electrode comprises a reference and a bridge electrolyte;

Fig. 3 is a schematic representation of a measurement setup for measuring a pH reference electrode against an external reference of the same type.

4 is a time-voltage diagram of an ideal measuring Ag / AgCl

Reference electrode against an external reference of the same type, with 3 mol / L KCl solution as the first component of the modifying electrolyte;

Fig. 5 time-voltage diagrams of an Ag / AgCl reference electrode against an external reference of the same type, wherein the measuring medium after 600 s sodium citrate was added and the reference electrode

a. a zirconia diaphragm having an average pore size of 70 nm or 120 nm;

b. a zirconia diaphragm having an average pore size of 200 nm;

c. a zirconia diaphragm having an average pore size of 400 nm or 800 nm;

Fig. 6 is a time-voltage diagram of an Ag / AgCl reference electrode to an external reference of the same design, the modifying electrolyte was mixed with 5x10 ^"4 mol / L sodium citrate;

Fig. 7 time-voltage diagram of an Ag / AgCl reference electrode whose

modifying electrolyte was added with 5x10 ^"4 mol / L sodium citrate, against an external reference of the same type, wherein the measuring medium after 600 s sodium citrate was added and the reference electrode

a. a zirconia diaphragm having an average pore size of 70 nm or 120 nm;

b. a zirconia diaphragm having an average pore size of 200 nm;

c. a zirconia diaphragm having an average pore size of 400 nm or 800 nm.

Figure 1 shows a schematic representation of a combined pH electrode with a pressurized reference electrode in section. The reference electrode has a substantially tubular housing 1, which is filled with a reference electrolyte 2 and in the wall of a transfer is arranged, which is here designed as a porous diaphragm 3 and through which the reference electrolyte 2 emerges continuously during operation. In the reference electrolyte 2 dives a first

Reference element or discharge element 4 a.

Furthermore, the pH measuring chain consists of a housing 1 arranged

Inner housing 5, which has a pH-sensitive glass membrane 6 and is filled with an inner buffer 7. In this inner buffer 7, a second discharge element 8 protrudes.

For pH measurement, the pH measuring chain dips into a measuring medium 9, so that

at least the glass membrane 6 and the porous diaphragm 3 are in contact with the measuring medium 9. The reference electrolyte 2 is in contact with the measuring medium 9 via the diaphragm 3.

One of the most well-known reference electrodes for pH measuring chains is the so-called Ag / AgCl electrode with a first diverting element 4 made of Ag / AgCl and one

chloride-containing first component of the reference electrolyte 2, for example, a KCl solution. Further known reference electrodes are silver chloride-free pH differential electrodes or redox reference electrodes.

According to the invention, the reference electrolyte 2, which essentially consists of a first component, is a surface-modifying component is added, so that the reference electrolyte 2 is the modifying electrolyte according to the invention. In silver ion-containing reference electrodes or reference systems, the reference electrolyte 2 is preferably a surface-modifying

Component which does not reduce silver ions even at temperatures above or above 130 ° C, such as citric acid and its salts. The modifying electrolyte of a pH differential electrode is preferably added with a surface-modifying component which itself constitutes a buffer system, such as a citrate buffer.

In addition, a variety of other types of reference electrodes for use in electrochemical sensors are known, which will not be described in detail here. Of course, it is also possible to modify these other types of reference electrodes according to the invention by adding a suitable surface-modifying component to the modifying electrolyte.

Furthermore, FIG. 1 shows the electrochemical potentials which occur during a measurement in a pH measuring chain and which can influence the measurement result. These include the potential E1 of the first diverter element 4, the diffusion potential or the diaphragm potential E2 through the diaphragm 3, the potential E3 of the second diverter, the potential E4 on the inside of the glass diaphragm 6, the asymmetry potential E5 between the first and second diverting element 4, 8 and the pH-dependent potential E6 on the outside of the

Glass membrane 6.

The potential occurring between the two diverting elements 4, 8, here the pH potential E5, can be measured with a voltmeter 13 and comprises the sum of the indicated potentials. This can be converted into a pH value. In a known Ag / AgCl reference electrode, the potential E1 according to Nernst's equation corresponds to the potential of the chloride ions on the first diverting element 4. The potential E1 is substantially constant as long as the concentration of chloride ions in the reference electrolyte 2 does not change. In known Ag / AgCl reference electrodes, this CI ^" concentration in the reference electrolyte is relatively constant at approximately c (CI ^" ) = 3 mol / L. This applies in particular to pressurized Reference electrodes, as a dilution or poisoning of the reference electrolyte 2 can be substantially avoided by the pressurization.

The diffusion potential E2 is a function of several quantities, to which the

Difference of the ion concentration between the reference electrolyte 2 and the measuring medium 9 and the surface charge of the diaphragm 3 belong, which is influenced by the zeta potential and the pore size of the diaphragm. The diffusion potential E2 should also be substantially constant and preferably have a value of 0 mV. The potentials E3 and E4 are substantially constant as long as the chloride ion potential at the second discharge element 8 and the H ⁺ ion potential of the inner electrolyte remain constant at the inner pH glass, which is due to the closed inner casing 5 with the inner electrolyte 7. The asymmetry potential E5 of a combined pH electrode is considered to be approximately constant as long as the temperature is constant. The potential E6 changes as a function of the H ⁺ potential on the outside of the glass membrane 6, that is, as a function of the pH of the medium to be measured.

FIG. 2 shows a combined pH measuring chain with a dual-chamber reference system. The pH measuring chain has essentially the same characteristics as the pH measuring chain according to FIG. 1, but this pH measuring chain has a different reference electrode. The first discharge element 204 is located in a further reference housing 220, in which the reference electrolyte 202 is arranged. This reference housing 220 has a further diaphragm 219, which represents a transfer between the reference electrolyte 202 and a bridge electrolyte 218 arranged in the housing 201. The bridging electrolyte 218 is in contact with the measuring medium 209 via the diaphragm 203. The bridge electrolyte 218 represents the modifying electrolyte in the sense of the invention and comprises a first component and a surface-modifying component, so that the diaphragm 203 is modified in-situ during operation upon passage of the bridge electrolyte 218.

The following examples illustrate the influence of modifying

Electrolyte added surface-modifying component to a pH measurement, especially in a protein-containing medium to be shown. Examples 1 to 7 were all carried out in an arrangement according to FIG. The arrangement shown comprises a pressurized reference electrode 312, which dips into a first vessel 31 1, and an external reference 315. The external reference 315 has a further vessel 310, wherein the two vessels 310, 31 1 via a diaphragm tube 314 with each other are connected. The diaphragm tube 314 is fluid filled and provides fluid communication between the two

Vessels 310, 31 1 ago. The vessel 310, for example a beaker, contains an electrolyte 316 of the external reference 315 in which a reference element 317 is immersed. The vessel 310 thus represents the half-cell of the external reference 315. The pressurized reference electrode 312 is immersed in a measuring medium 309 arranged in the second vessel 31 1. The reference half cell or reference electrode 312 comprises a housing 301, which is filled with a reference electrolyte 302 and has a diaphragm 303 as a transition to the measurement medium 309. A first diverting element 304 in turn dips into the reference electrolyte 304. The

Reference electrode 312 is pressurized so that the reference electrolyte 302 can continuously leak through the diaphragm 303.

In order to demonstrate the influence of the addition of a surface-modifying component, such as sodium citrate, on the measurement properties of a reference electrode, measurements with differently designed reference electrodes 312 were initially different with the arrangement according to FIG

Compositions of reference electrolyte 302 and different

Measuring media 309 performed.

Example 1: Standard reference electrode against an external reference

As the reference electrode 312, a pressurized Ag / AgCl electrode having an Ag / AgCl wire as the first diverter 304 and a microporous zirconia diaphragm 303 was used. The reference electrolyte 302 included only a 3 mol / L KCI aqueous solution as a first component and no surface-modifying component. The reference electrode used was

pressurized to ensure a controlled passage of the reference electrolyte 302 through the diaphragm 303. As an external reference, the vessel 310 was used which contained an Ag / AgCl wire as the second dissipation element 8 and an aqueous 3 mol / L KCl solution as the electrolyte 317.

The measuring medium was likewise an aqueous 3 mol / L KCl solution and the two vessels 310, 31 1 were connected to one another via a large-pore diaphragm tube 314.

Since, in this example, all the electrolytes as well as the measuring medium were aqueous KCI solutions of the same concentration and thus could not form a concentration gradient via the diaphragm 303 or the diaphragm tube 314, in one measurement the diffusion potential E2 was essentially constant zero and thus the sum of all potentials E1 to E6 is substantially equal to zero.

FIG. 4 shows a plot of the time-voltage diagram of the reference electrode against an external reference, which reproduces this situation. The plot shows that the measured voltage over time was essentially at about zero. Example 2: Citrate-containing measuring medium

In a further example, which corresponds essentially to Example 1, after a measuring time of 600 s, the measuring medium 309, a 3 mol / L KCl solution, was mixed with 0.1 mol / l sodium citrate.

These measurements were made with reference electrodes 312 with different

Pore sizes of the porous zirconia diaphragm 303 performed, more precisely, with average pore sizes of 70nm, 120nm, 200nm, 400nm and 800nm.

When sodium citrate is added to the measuring medium 309, first the surface of the diaphragm 303 is wetted with citrate ions, whereupon the surface potential (zeta potential) and the isoelectric point (ISE) of this surface change accordingly so that also the diffusion potential E2 changed, that is no longer constant. The change in the diffusion potential E2 is particularly pronounced for small-pore diaphragms and in measuring media with low ion concentration and resulting strong diffusion.

Due to the change in the potential E2, the sum of the potentials and thus the result of the measurement changes, as it does as a potential jump in the

Diagrams 5a-c can be seen, wherein the observed tendency of the diagrams is independent of the diaphragm pore size used. In FIG. 5 a, the solid line shows the course for a diaphragm with an average pore size of 70 nm and the dashed line the course for a diaphragm with an average pore size of 120 nm. FIG. 5 b shows the course for a diaphragm with an average pore size of 200 nm and shown in Figure 5c for diaphragms with an average pore size of 400 nm (solid line) and 800 nm (dashed line).

With small pore diaphragms the effect is stronger (FIG

Probability increases that an ion (including hydration shell) with the surface of the diaphragm 303 interacts or adsorbed on this and / or retained by the diaphragm 303, resulting in a charge separation and thus a change in the diffusion potential can result.

In media with low ion concentration, this effect is stronger, as the driving force for diffusion and thus diffusion due to the

Concentration difference of the ions between reference electrolyte 302 and

Measuring medium 309 increases.

The greater the likelihood that an electrolyte ion will interact with the diaphragm surface, the greater the noise of the measurement signal and the change in the diffusion potential E2. This probability increases with decreasing pore size in the diaphragm. The change in the diffusion potential E2 leads to the potential jump after 600 s and thus to a measurement error in the combined pH electrode. Example 3 Citrate-containing reference electrol

In this example, as the reference electrode 312, a pressurized Ag / AgCl electrode was used with Ag / AgCl wire as the first diverter 304 and a

Diaphragm 303 made of porous zirconia used. The reference electrolyte 302 consisted of an aqueous 3 mol / L KCl solution as the first component, which was added with sodium citrate in a concentration of c (Na citrate) = 5x10 ^"4 mol / L as a surface-modifying component, so that the reference electrolyte 302 as The reference electrode used was pressurized to ensure a controlled passage of the reference electrolyte 302 through the diaphragm 303.

As an external reference electrode, the first vessel 310 was used, which contained an Ag / AgCl wire as reference element 317 and an aqueous 3 mol / L KCl solution as electrolyte 316.

The measuring medium was an aqueous 3 mol / L KCl solution. For this example, the potential E2 is substantially constant, but deviates from zero, as it is a function of the diaphragm pore size which has been altered by occupancy with citrate ions. Thus, the sum of the potentials is a constant, deviating from zero size, as can be seen in the diagram of Figure 6. The in situ occupancy or modification of the diaphragm surface by the preferably continuously flowing out surface-modifying component, here sodium citrate, leads to a change in the reference potential, which, however, can be compensated by a suitable calibration. The zero point shift of the combined pH measuring chain to be recognized in FIG. 6 can also be adjusted or set to zero again by adapting the inner buffer 7. Example 4 Citrate-containing reference electrolyte and citrate-containing measuring medium

In this example, both the reference electrolyte 302 and the

Measuring medium 309 mixed with sodium citrate. For this purpose, the aqueous 3 mol / L KCl solution of the measuring medium 309 was admixed with 0.1 mol / l sodium citrate after a measuring time of 600 s. The other parameters of the arrangement corresponded to Example 3, wherein here measurements were carried out with different pore sizes of the porous zirconium oxide diaphragm 3, more precisely with pore sizes of 70 nm, 120 nm, 200 nm, 400 nm and 800 nm.

The diaphragm surface was, as in Example 3, in-situ and preferably continuously occupied by the nachströmenden due to the pressurization of the reference electrode 312 reference electrolyte 302 with citrate ions. Therefore, the diaphragm surface was already occupied when the citrate salt was added to the measurement medium, so that the surface charge and the diffusion potentials of the diaphragm changed only insignificantly. As the diagrams of FIGS. 7a-c show, after citrate addition to the

Measuring medium no potential jump to recognize more. This effect is again independent of the pore size of the diaphragms used. The still existing zero deviation can again be easily compensated by changing the inner buffer. In FIG. 7a, the solid line shows the course for a diaphragm with an average pore size of 70 nm and the dashed line the course for a diaphragm with an average pore size of 120 nm. FIG. 7b shows the course for a diaphragm with an average pore size of 200 nm and shown in Figure 7c for diaphragms with an average pore size of 400 nm (solid line) and 800 nm (dashed line). By means of examples 1 to 4 it could be shown that disturbances of the

Measurement result of a pH measuring chain in citrate-containing measuring media can be corrected by addition of citrate salt as a surface-modifying component to the reference electrolyte. Furthermore, it has been shown that even a low concentration of citrate in the modifying electrolyte, in this case the reference electrolyte, is sufficient to add the diaphragm surface in situ and preferably continuously modify and thus counteract further attachment of citrate from the measured medium.

Example 5: Differential electrode with reference electrode of the invention

In a further embodiment, the measurement setup was adapted according to Example 4 by using a pH reference electrode instead of an Ag / AgCl reference electrode. There were as in Example 4

Measure reference electrodes with different pore sizes of the diaphragms. The reference electrolyte used was a pH 4.6 buffer solution containing ascorbic acid as the surface-modifying component at a concentration of about 0.05 mol / L.

As already described in connection with Example 4 after 600s

Measuring time the medium a salt of the surface-modifying component, here sodium ascorbate added.

The addition of ascorbic acid to the reference electrolyte, here a pH buffer system, also showed no potential jump even after addition of the ascorbate to the measurement medium, the effect being independent of the pore size of the diaphragms used. The still existing ones

Zero offset can in turn be easily compensated by changing the inner buffer. Example 6: Use of Metallic Structures as Diaphragm

Analogous to the measurement setups used in Examples 1 to 4, also measuring structures with metallic structures, more precisely platinum wire bundles as

Diaphragm realized and measured. It was noticeable in the measurements carried out that new platinum wire bundles had a relatively large pore size, whereby reference electrodes 312 with such new platinum wire bundles showed the same effects as the reference electrodes with large-pore zirconia diaphragms from Examples 1 to 4. Platinum wire bundles have the property to be very soft and tend to deformation during mechanical cleaning and mechanical action. The pores in the wire bundle become increasingly smaller and diffusion potential effects greater. After repeated mechanical cleaning of the platinum wire bundles, for example by grinding or heavy abrading of the diaphragms, the potential effects of the diaphragms became ever greater, so that finally similar results were achieved, as with the microporous diaphragms (70 nm, 120 nm) from Examples 2 to 4. Likewise could the diffusion potential effect by using citrathaltiger

Reference electrolytes are reduced in a similar manner as in Example 4.

From these results, it could be concluded that diaphragms made of metallic structures are less susceptible to the adsorption of foreign substances by the addition of a surface-modifying component to the reference electrolyte.

Example 7: Measurement in protein-containing measuring media

In a further embodiment, pH measuring chains with different reference electrodes for determining the pH of a

protein-containing medium used and their performance compared. These comparative measurements were made in a "mammalian bioprocess" for the production of recombinant lactogen proteins from ovarian cells of

Hamsters (CHO = Chinese hamster ovary) performed.

In this bioprocess occur as proteins u. a. bovine serum albumin (BSA) with an isoelectric point of about 4.5 and the milk protein casein with a

isoelectric point of about 4.6. For the bioprocess, a 2.5 L bioreactor with a working volume of 2 L was used (MBR Bioreactor, Zurich). Cell growth of CHO cells was performed in a reaction medium with 7 g / L glucose and 7 mmol / L glutamine as the primary carbon source at a temperature of 36.5 ° C. The oxygen saturation was adjusted to 50% and kept at this value by gassing with nitrogen and / or oxygen. The pH of the biomass was adjusted to pH 6.8 and kept at this value by metering 0.3 mol / L NaOH. The biomass was then seeded with 8.5x10 ⁶ CHO cells / mL and maintained by continuous thinning and loss of the cells at 10x10 ⁶ cells / mL. The throughput rate was about 1.5 bioreactor volume / day and the reactor was operated continuously for seven days.

The bioprocess was monitored with different sensors, u. a.

Oxygen sensors, temperature sensors and CO ₂ sensors, as well as several pH electrodes of different design. After completion of the seven-day cell production, the measurement performance of the different pH electrodes was compared.

The following pH measuring chains were used:

^* The concentrations given are based on pH 7 and given in mol / L.

Two pH measuring chains of the type PH-3 were used, with one as

process-controlling pH measuring chain was used.

After completion of the seven-day bioprocess, the result was as follows regarding the measurement performance of the different pH electrodes. Measuring chain PH-1 showed a strongly fluctuating pH value during the bioprocess and no pH value at the end of the bioprocess. The pH values displayed by the PH-2 electrode were subject to a high level of noise of up to ± 1 pH units, but in contrast to PH-1, a pH value could still be read after the process had ended. During the bioprocess, the pH chain PH-3 showed a stable value of pH 6.8 +/- 0.1.

The diaphragms of the reference electrodes of the measuring chains PH-4 and PH-5 included an old, highly depressed platinum wire bundle and therefore had a very small pore size. Measuring chain PH-4 behaved analogously to PH-1. The displayed pH fluctuated strongly and could not be read at the end of the bioprocess. Measuring chain PH-5 as well as PH-3 showed the correct value of pH 6.8, but with a somewhat stronger noise of 0.2 pH units.

After completion of the bioprocess, all five pH chains were included

Standard buffer solutions calibrated at pH 4 and pH 7. The calibration showed that the measuring chains PH-1 and PH-4 could not be calibrated due to insufficient final values due to excessive noise.

Calibration of pH chain PH-2 could just be carried out, but only after long waiting times, as the response time of the pH chain PH-2 had slowed down considerably. It was found that the slope of the measuring chain PH-2

had declined and this indicated in particular because of the long response time and / or the strong noisy signals not sufficiently stable end values.

An attempt was made to regenerate and recalibrate the PH-1, PH-2 and PH-4 electrodes. For this, the measuring chains were in a 12 h

Regenerated hydrochloric acid / pepsin-containing regeneration solution (Mettler-Toledo) and re-calibrated in pH 4 and pH 7 buffer solutions.

Measuring chain PH-2 could be regenerated in this way and then recalibrated. After this procedure, the zero point of PH-2 was 5 mV with a slope of 97.6%. The measured values of the measuring chains PH-1 and PH-4 after regeneration during the calibration in buffer pH 4 still showed a strong noise of +/- 5 mV and took a very long time for the measured value determination. After changing to buffer pH 7, the measured values started to strongly roar again and no final value could be determined.

Obviously, even a twelve-hour regeneration of the chains in hydrochloric acid / pepsin was not sufficient to increase protein absorption at the surface of the protein

Completely eliminate diaphragms.

Even after twelve hours of regeneration, the measuring chains PH-1 and PH-4 showed neither sufficient measuring performance nor sufficient reproducibility in protein-containing measuring media and therefore could no longer be used for further measurements.

Although measuring chain PH-2 could be regenerated, it showed a significantly reduced measuring performance in protein-containing solutions. On the other hand, the measuring chains PH-3 PH-5, which an inventive

Reference electrode with a modifying electrolyte, a very good calibration behavior and the final values came quickly. Even after seven days of use in a high protein bioprocess, they showed

Measuring chains still steepnesses of 98% (PH-3) and 96.3% (PH-4). Although the invention has been described by way of illustrating specific embodiments, it will be apparent that many more

Embodiments in knowledge of the present invention can be provided, for example by combining the features of the individual embodiments with each other and / or individual functional units of

Embodiments are exchanged. List of Reference Numerals, 201, 301 Housing

, 202, 302 reference electrolyte

, 203, 303 Diaphragm

, 204, 304 first diverting element

, 205, 305 inner housing

6, 206 glass membrane

7, 207 inner buffer

8, 208 second discharge element

, 209, 309 Measuring medium

310 first vessel

31 1 second vessel

312 reference electrode

, 213, 313 voltmeter

314 Diaphragm tube

315 external reference

316 electrolyte

317 reference element

218 bridge electrolyte

219 further diaphragm

220 reference housing

E1 potential of the first diverting element

E2 diffusion potential / diaphragm potential

E3 potential of the second diverting element

E4 potential of the inside of the glass membrane

E5 asymmetry potential

E6 potential of the outside of the glass membrane

Claims

claims

1 . Reference electrode with at least one housing (1, 201, 301), a first discharge element (4, 204, 304), a modifying electrolyte, which is flowable and arranged in the housing (1, 201, 301), and a porous diaphragm (3, 203, 303) which provides fluid communication between the modifying electrolyte and a measuring medium (9, 209, 309) and through which the modifying electrolyte emerges during operation, the modifying electrolyte comprising a first component and a surface modifying component forming the surface of the porous

Diaphragm (3, 203, 303) modified in situ during the passage of the modifying electrolyte.

2. Reference electrode according to claim 1, characterized in that the

Surface of the porous diaphragm (3, 203, 303) is continuously modified.

3. Reference electrode according to claim 1 or 2, characterized in that it is pressurized, so that the modifying electrolyte during operation

continuously through the porous diaphragm (3, 203, 303) emerges.

4. Reference electrode according to one of claims 1 to 3, characterized in that the modifying electrolyte is a reference electrolyte (2, 302), in which the first diverting element (4, 304) is immersed.

5. Reference electrode according to one of claims 1 to 3, characterized in that the modifying electrolyte is a bridge electrolyte (218) and that the reference electrode further comprises a reference electrolyte (202) and a

Reference housing (220) with a further diaphragm (219), wherein the first diverter (204) immersed in the reference electrolyte (202) and the reference electrolyte (202) via the further diaphragm (219) with the

Bridge electrolyte (218) is in contact.

6. Reference electrode according to one of claims 1 to 5, characterized in that the surface-modifying component comprises an electrostatically acting substance.

7. Reference electrode according to one of claims 1 to 5, characterized in that the surface-modifying component is a sterically demanding

Substance includes.

8. Reference electrode according to one of claims 1 to 7, characterized in that the surface-modifying component comprises an organic substance which has at least one hydroxy substituent and at least one carbonyl substituent.

9. Reference electrode according to claim 8, characterized in that the

surface-modifying component comprises lactic acid, citric acid, malic acid, tartaric acid, ascorbic acid, their salts or mixtures thereof.

10. Reference electrode according to claim 8, characterized in that

the surface-modifying component comprises polylysine-polyethylene glycol.

1 1. Reference electrode according to one of claims 1 to 10, characterized in that the modifying electrolyte, the surface modifying component in a concentration of less than 0.1 wt.%, In particular from about 0.05 to 0.1 wt.%, Has.

12. Reference electrode according to one of claims 1 to 1 1, characterized in that the porous diaphragm (3, 203, 3033) substantially comprises a ceramic material.

13. Reference electrode according to one of claims 1 to 1 1, characterized in that the porous diaphragm (3, 203, 303) substantially comprises a metallic structure. Method for the in situ modification of a porous diaphragm (3, 203, 303), which is arranged as a fluid connection to a measuring medium (9, 209, 309) in a reference electrode according to one of the preceding claims, the method comprising the following steps:

a. Adding a surface-modifying component to a modifying electrolyte disposed in the reference electrode; and b. Ensuring the exit of the modifying electrolyte through the porous diaphragm (3, 203, 303).

A method according to claim 14, characterized in that the modifying electrolyte in operation continuously through the porous diaphragm (3, 203, 303) emerges.