WO1985004482A1

WO1985004482A1 - Electrochemical assembly

Info

Publication number: WO1985004482A1
Application number: PCT/GB1985/000101
Authority: WO
Inventors: Peter Halling
Original assignee: University Of Strathclyde
Priority date: 1984-03-27
Filing date: 1985-03-15
Publication date: 1985-10-10
Also published as: EP0175732A1; GB8407835D0; IT8567294A0

Abstract

An electrochemical assembly (30) for continuously monitoring the level of ammonia in a fermenting medium comprises a housing (14) incorporating a gas-permeable ion-impermeable outer membrane (21) which is capable of transmitting ammonia from the medium into the housing (14). A monitor electrode is located within housing (14) and incorporates a monovalent cation-sensitive-glass inner membrane (19) the latter being separated from outer membrane (21) by a thin aqueous film (20) of electrolyte solution which is pH buffered. A reference electrode (16) is provided within housing (14) and an electrical connection (17) is provided between the film (20) and the reference electrode (16).

Description

ELECTROCHEMICAL ASSEMBLY

This invention relates to an electrochemical assembly for continuously monitoring the level of ammonia in a fermenting medium.

In many microbial processes such as occur in industria fermentations the presence and concentration or activity level of ammonia and ammonium ions is of practical significance. It is therefore desirable to monitor continuously the level of ammonia since this provides a measure of the concentration or activity level of ammonia and ammonium ions in the medium concerned. Known apparatus for this purpose has the disadvantage that it is also sensitive to the presence of substances such as carbon dioxide, volatile amines, acidic or basic volatile species, and monovalent cations such as the potassium ion, at least one or more of which is present in variable but significant concentrations in most fermenting media.

It is an object of the present invention to provide a new and improved form of electrochemical assembly for continuously monitoring the level of ammonia in a fermenting medium.

According to the present invention there is provided an electrochemical assembly for continuously monitoring the level of ammonia in a fermenting medium, said assembly comprising a housing having a portion in the form of a gas- permeable ion-impermeable outer membrane for transmitting into said housing ammonia from said medium, a monitor electrode located within said housing and comprising an electrode container having a portion in the form of a monovalent cation-sensitive-glass inner membrane, a reference electrode located within said housing, a body of aqueous electrolyte solution containing a pH buffer located within said housing and forming a thin aqueous film in areal contiguity with both of said membranes, and means forming an electrical connection between said body of aqueous electrolyte solution and said reference electrode. the assembly being sufficiently robust as to be capable of withstanding sterilisation by water and/or steam under pressure at temperatures of 115 C and higher.

It will be understood that the inner membrane by virtue of being of the monovalent-cation-sensitive-glass type is sensitive to the limited range of monovalent cations which are physically small, such as sodium, potassium, lithium, hydrogen and ammonium ions. Accordingly volatile species, although traversing the outer membrane, are in- capable of producing cations in the thin aqueous film to which the monitor electrode is sensitive. As regards ammonia and ammonium ions in the fermenting medium ammonia gas traverses the outer membrane and in the thin aqueous film forms ammonium ions which alter the electrochemical potential of the monovalent-cation-sensi ive glass inner membrane and this is measured electrically relative to the reference electrode.

It will be understood that in fermenting media and aqueous solutions ammonia and ammoniu 'ions co-exist in relative proportions determined by the pH of the media

(or solution) and because the body of aqueous electrolyte solution within the housing of the assembly of the present invention is pH buffered (at a predetermined level) measurement of the ammonium ion therein (as previously explained) provides also a measure of the ammonia therein and under equilibrium conditions the concentration or activity level of the ammonia in the aqueous electrolyte body is the same as that in the fermenting medium. Furthermore, if the pH of the fermenting medium is known, as is usually the case, the respective concentrations of ammonia and of ammonium ion in the fermenting medium can be easily derived.

The outer membrane may be made of silicone rubber or teflon and may have a thickness in the range 5 to 100^_ι_ra. A thickness in the range 10-20.um is preferred in order to provide sufficient mechanical strength whilst maintaining sufficiently small response time. The outer membrane may be mechanically supported over its external face by open meshwork. The inner membrane may be made of a glass having the composition 27% Na₂0; 5% Al₂0₃; 68% Si0₂ and may have a thickness in the range 0.05 to 1.00 mm although a thickness in the range 0.2-0.6 mm is preferred. Conveniently the surface of the inner membrane which is in areal contact with the thin aqueous film is either flat (planar) or convex towards the outer membrane.

The body of aqueous electrolyte solution provides electrical conductance by the presence of ions therein. The preferred anion is chloride ion and preferred cations include tetramethylammonium and magnesium ions. The preferred pH buffer species forming part of the body of • aqueous electrolyte are non-volatile organic amines, phenols, borate, or carbonate bicarbonate ions. It will of course be understood that the content of the body of aqueous electrolyte must not adversely affect operation of the inner membrane or in the reference electrode or in the means forming the electrical connection between the electrolyte body and the reference electrode.

The means forming the electrical connection between the electrolyte body and the reference electrode may be a liquid junction in the form of a porous plug in an otherwise impermeable wall separating the reference electrode from the electrolyte body.

The reference electrode may conveniently be an

++ Ag/AgCl rod immersed in a solution of 1M Mg -glycine buffer pH9 containing 3M MgCl₂.

The monitor electrode may conveniently be an Ag/AgCl rod immersed in a solution of 0.1M KC1.

The outer membrane itself constitutes a sterility barrier preventing contaminants entering the fermenting medium via the interior of the electrode assembly, but, preferably the assembly additionally comprises means forming a sterility barrier around said body of electrolyte solution and within said housing to prevent contaminants entering said body so that in the event of mechanical failure of the thin outer membrane contamination of the fermenting medium is prevented.

An embodiment of the present invention will now be described by way of example with reference to the accompanying drawing, in which:

Fig. 1 is a cross sectional view of an electrochemical assembly according to the present invention; and

Fig. 2 is an enlarged view of a detail of Fig. 1. In the drawing the assembly 30 comprises a generally cylindrical stainless-steel housing 14 across the lower open end of which is located an outer membrane 21 made of. silicone rubber, membrane 21^' being held in place by a rubber 0-ring 18 and a threaded collar 9 threadedly engaging housing 14. Membrane 21 is gas-permeable and ion-impermeable as previously explained.

Within housing 14 there is located a monitor electrode comprising a container in the form of a generally cylindrical glass tube 3 the lowermost end of which is closed by the inner membrane 19. Membrane 19 is formed of monovalent- cation-sensi ive glass having a formulation 27% Na_0; 5% Al_0₃; 68% Si0₂ whereas tube 3 is made of an ion- insensitive glass such as a borosilicate. The glasses of tube 3 and membrane 19 are joined by an annular joint 22 comprising five annular zones of intermediate glass composition for the purpose of providing a gradual transition between the two glass forms so that stresses due to differential thermal expansion are constrained to a tolerable level for the purpose of withstanding sterilisation. Within container tube 3 is a body of 0.1M KCl solution 6 immersing electrode rod 15 which is Ag/AgCl. Also within housing 14 there is located a reference electrode comprising glass tube 3A concentrically surrounding tube 3 and united with tube 3 at the lower end of tube 3A so as to form a container for a solution

++ 7 which is 1M Mg -glycine buffer pH 9 containing 3M MgCl,.

Solution 7 immerses rod electrode 16 which is Ag/AgCl.

At the upper end of the assembly 30 tube 3 is closed and provides a glass seal for exit of rod 15 which is provided with an electrically insulating plastic sleeve 11 the exterior of which has a conductive screen 12 connected directly to rod 16. The upper end of glass tube 3A forms a glass seal for components 10,11 and 12 and externally of the glass seal a plastic seal 13 is provided for integrity. Tube 3A at its upper end incorporates an integral collar against which a rubber

0-ring 2 seats being held in place by a threaded collar 1 which threadedly engages, the uppermost end of housing 14. By this method inner membrane 19 is biassed towards outer membrane 21 and by virtue of the axial lengths of housing 14 and tubes 3, 3A being matched inner membrane 19 is capable of engaging outer membrane 21 and causing limited stretch therein. For this reason it is preferred that the surface of inner membrane 19 proximal outer membrane 21 is either flat or slightly convex (towards membrane 21) . In accordance with an important aspect of the present invention a body 8 of aqueous electrolyte solution which is pH buffered is provided within the annular space between housing 14 and glass tube 3A so that the region between the membranes 19, 21 is flooded with this solution and, when top collar 1 is appropriately adjusted, a thin aqueous film 20 is formed between membranes 19, 21 being in areal contiguity with both membranes 19, 21. The body 8 has the same composition as the solution 7 and in conjunction with porous plug 17 in the wall of tube 3A provides an electrical connection between the reference electrode and the thin film 20. Furthermore, in order to provide a sterility barrier preventing contamination entering the top of housing 14 penetrating downwardly and possibly out into a fermenting medium via a ruptured outer membrane 21 a silicone rubber 0-ring seal ^"is provided between housing 14 and glass tube 3A above the body 8 of aqueous solution.

The assembly 30 is constructed as has previously been explained to provide a measure of the ammonium ion level within aqueous fil 20 arising from the diffusion through outer membrane 21 of ammonia gas, such gas being present in a fermenting medium. The constructional details of the assembly 30 will be better understood in terms of quantita¬ tive analysis of its response at equilibrium of the diffusing gas (NH^) , that is when the concentration or activity level of the ammonia on either side of the outer membrane 21 is equal.

In an aqueous solution the equilibrium condition is given by ^κa " [^NH3. [^H+] (1)

where Ka = dissoci Kati.on constant of NH4..

Using the symbol pX to denote - log,_QX equation (1) becomes pNH, = pK + pNH, - pH and since pNH___ and pKa have the same value on either side of the outer membrane 21 it follows that

(pNH₄)₀ = (pNH₄)_I + (pH)_Q - (pH_#I (2) where the subscripts 0 and I respectively denote outside the assembly (i.e. in the fermenting medium) and inside the assembly (i.e. in the aqueous film 20) . The electrical potential of the monitor electrode is linearly related to (pNH. )._j. within the normal operating range of the inner membrane 19 whilst (pH) is maintained constant by the buffer within aqueous film 20 and (pH) _ is separately measurable (and may even be controlled at a' constant level) so that by measuring (pNH.)- a measure of (pNH.)₀ is also provided.

The response rate of the assembly 30 is derivable from the equation given by J.W. Ross et al (Pure and Applied Chemistry, Vol. 36, pages 473-487 published 1973), namely

, , ^dCB . d .log 6 _ -D.K (3) dC dt ^{e ~} Z. where C = concentration of diffusing gas in film 20;

C___> = concentration of the bound form of the gas in film 20 £ = fractional deviation from equilibrium relative to the final equilibrium concentration in film 20; D = diffusion coefficient of the gas in the outer membrane 21; K = partition coefficient of the gas into membrane 21; I - thickness of film 20; m = thickness of membrane 21; and t = time

It can be shown that for the assembly described, because ^dCB . ₁₀(pK_a - (PH)_∑ ⁾ _{= constant} dC the response time T is given by

where ΔC = change in gas concentration in the medium;

C- = final gas concentration after the ΔC change in the medium; and 6. =.the fractional deviation taken to define complete response.

By way of example if I - 10 yum; m = 20 ,um; D.K = 10

2 cm /s (i.e. silicone rubber); pK_a = 9.3; (pH)_ = 9.0; ^β = 0.01; __-C/C_f = 0.2; it follows that T = 240 seconds, a value which is acceptable for monitoring and control of fermenting media. The choice of pH level for the film 20 is governed by two factors. Firstly, equation 4 shows that minimum- response time is achieved by maximising (pH)_. Secondly, the sensitivity of the inner membrane 19 to H. generally requires (pNH.)_ 6 to obtain a useful signal. For a typical fermenting media where (pNH.)- = 3 and (pH)_ = 6 it can be shown from equation 2 that (pH)_ must be less than or equal to 9. Therefore (pH)_ = 9 is the preferable value. The assembly 30 by virtue of having its components made of stainless steel, glass, silicone rubber and/or teflon in the construction shown in the drawing is sufficiently robust to be capable of withstanding sterilisation by water and/or steam under pressure of the order of 1 or 2 atmospheres at temperatures of 115 C up to 150 C. Furthermore the.various electrolyte solutions are either relatively volatile .and held within a pressure vessel formed within the assembly 30 or relatively non¬ volatile so that there is no loss of electrolyte during sterilisation. Additionally because collar 1 threadedly engages the uppermost end of housing 14 release of this collar permits tube 3A to be moved axially away from outer membrane 21 which enables sterilisation to be effected with minimum stress on outer membrane 21.

Claims

1. An electrochemical assembly for continuously monitoring the level of ammonia in a fermenting medium, said assembly (30) being qharacterised by the combination of a housing (14) having a portion in the form of a gas-permeable ion- impermeable outer membrane (21) for transmitting into said housing (14) ammonia from said medium, a monitor electrode located within said housing (14) and comprising an electrode container (3) having a portion in the form of a monovalent cation-sensitive-glass inner membrane (19) , a reference electrode (3A,7,16) located within said housing (14) , a body (8) of aqueous electrolyte solution containing a pH buffer located within said housing (14) and forming a thin aqueous film (20) in areal contiguity with both of said membranes (19,21) , and means (17) forming an electrical connection between said body (8) of aqueous electrolyte solution and said reference electrode (3A,7,16), the assembly (30) being sufficiently robust as to be capable of withstanding sterilisation by water and/or steam under pressure at temperatures of 115 C and higher.

2. An assembly as claimed in claim 1, characterised in that the outer membrane (21) is made of silicone rubber or teflon.

3. An assembly as claimed in claim 2, characterised in that the outer membrane (21) has a thickness in the range 5 to 100 μm.

4. An assembly as claimed in any preceding claim, characterised in that the outer membrane (21) is mechanicall supported over its external face by open meshwork.

5. An assembly as claimed in any preceding claim, characterised in that the inner membrane (19) is made of glass.

6. An assembly as claimed in claim 5, characterised in that the inner membrane (19) has a thickness in the range 0.05 to 1.00 mm.

7. An assembly as claimed in any preceding claim, characterised in that the surface of the inner membrane

(19) which is in areal contact with the thin aqueous film

(20) is either flat or convex towards the outer membrane (21).

8. An assembly as claimed in any one of claims 5-7, characterised in that the electrode container (3) is made of glass and the inner membrane (19) is joined to the electrode container (3) by a plurality of annular zones (22). of intermediate glass composition providing a gradual transition between the glass forms of inner membrane (19) and the container (3) .

9. An electrochemical assembly for continuously monitoring the level of ammonia- in a fermenting medium substantially as hereinbefore described with reference to the accompanying drawing.