CA2184954A1

CA2184954A1 - Reference electrode assembly

Info

Publication number: CA2184954A1
Application number: CA002184954A
Authority: CA
Inventors: Andy D. C. Chan; Joseph S. Foos; James E. Rasmussen; Richard L. Schulkind; John A. Zalenski
Original assignee: Ciba Corning Diagnosys Corp
Current assignee: Bayer Corp
Priority date: 1995-11-03
Filing date: 1996-09-06
Publication date: 1997-05-04
Also published as: US20040188252A1; US6719888B1; KR970028540A; EP0772041A1; EP0772041B1; MX9603915A; DE69628895D1; ATE244400T1; PL316056A1; DK0772041T3; ES2201156T3; AU6084496A; JPH09170998A; DE69628895T2

Abstract

A reference electrode assembly having a constrained-diffusion liquid junction between a liquid junction solution and a sample solution having separate flow paths is disclosed. The assembly consists of a flow cell having attached to it a constraint comprising a region of porous material permeable to water and salts, a remote reservoir for holding a liquid junction solution, a means for moving the liquid junction solution from the reservoir to the constraint, and a reference contact region. Such a reference electrode device is useful in pH and/or ion selective electrode potentiometric sensors and is suitable for use in a mini-integrated electrochemical analyzer.

Description

~ 2 1 849~4 Reference Electrode Assembly ~ield of Invention This invention provides a reference electrode device of the col~ll~illed-diffusion liquid junction type useful in pH and/or ion-selective electrode (ISE) s potentiometric sensors and is particularly suitable for use in a mini-integrated electrochemical analyzer.
Background of Invention Conventional types of reference electrodes have a liquid junction where the sample meets the junction solution. The junction is typically either open or 0 co~ ained. In an open junction system, the liquid junction operates by free diffusion. In a conslldilled-diffusion junction system, a region of porous m~teri~l permeable to water and salts (a membrane, porous plug, frit, or the like) is placed at the site of the liquid junction. The porous m~teri~1 acts as a co~ t whereby - pds:~e of large molecules (such as protein) and bulk liquid l~ oll is generally lS hindered.
The liquid junction solution (also commonly referred to as the salt bridge solution) typically COIll~inS a solution s~ ~l with a salt (such ~ an e.~ f~rent salt,in~ 1ingKCl, KNO3)whichfunctionstoreduceand"~ ;"col.~.Ithe int~rf~ sl pot~ ial which develops across the liquid junction boulld~ ~, typically 20 l~f~.le;l to as a liquid junction potential. The dilr.,r~.lce in liquid junction potPnt ~ the system calibIator and the sarnple is l~,fe.l~ to as the residual liquid junction pot,.lLal. Typically, the residual liquid junction potential illCl~S ~ the ionic :~ ~lh LLf~nce bc lween the system calibrator sollltiQn and the sample sol1~tion i~c,e ases. The residual liquid junction potential is ~en~r~11y con~;~lered to - r~ 2184954 ~3 colllprolllise the accuracy of the associated potentiometric sensors and therefore a multi-use reference electrode is typically designed to ...i~ the residual liquid junction potential for as long as possible, while balancing other design constraints.
In potentiometric systems that are designed with mini~tl-ri~d working 5 electrodes (typically pH and/or ion selective electrodes), the necessity of the junction solution makes mini~ lion of a reference electrode difficult. Further, for the lef~ellce electrode to have a multiple use capability, the liquid junction solution must be present in a volume and concentration to minimi7~ the residual liquid junction potential over its useful lifetime. In order to minimi7P errors due to the residual liquid lo junction potential, the junction solution is generally salu,~led with the e.lL~il.~lsferent electrolyte salt.
Conventional reference electrodes utilize a fefe.ellce contact region (also sometimes lcr~ d to as electrode elements) immersed in a st~gn~nt junction solution which collk~ s a col~ l concentration of the e luill~lsferent salt. The reference 15 contact region is often silver based, c~n~i~ting of an electrochemically reversible redox electrode couple such as AgtAg+ and Ag/AgCl. When salt solutions are used with silver based ,~r~ ce contact regions such as Ag/AgCl, the AgCl is ~usce~tible to dissolution. In co~1,~ed-diffusion type liquid junctions, this ~licsollltion iS
~loblc~tic because it leads to subsequent ~l~;pi~ on of silver salts on the region of 20 porous m~tPri~l col.~l.,-;..1 thus leading to undesirable fouling ofthe co~l.iillt, which in turn generally results in an erratic reference electrode l,f . r~....A,.~e Cornmonly, bec~ ~se ofthe above-described fouling problem ~soci~te~l with the use of silver based l~,f~llce contact regions, barrier ~ .kl --~PS have been used to reslrict Ag+ ion mi~tion from the reference contact region to the porous m~tPri~l f~ 2 1 84954 ~3 constraint region. The use of a barrier membrane, however, carries with it inconvenience because the first use wet-up of the reference electrode is hindered by the barrier membrane, thus requiring a long soaking time in the junction solution prior to the first use.
s Another common problem associated with the use of saturated e~luill~lsferent salt solutions in consll~ ed-diffusion type of liquid junction reference electrodes is that when the refc.e1lce electrode is stored and/or used at sub-ambient t~ e1~lules, salt cryst~lli7~tion and p1ecipil~lion may occur between the reference contact region and the region of the porous m~t~ri~l at the junction, which in turn leads to erratic 1efe.~ ,lce electrode potentials. An additional problem associated with the use of a s,llul~t~d e luill~lsferent salt solution is that the s~lulaled solution may contribute to reproducibility and/or accuracy problems with blood samples because of i,lt~.re1ence caused by p1ccipil~lion of protein and crenation of red blood cells present in the sample.
R~s~ . ~oh:~ for holding of the junction solutions have been described for open (free diffusion) junction type of 1~;r~,.c.lce electrodes, where there is no region of porous m~tP~l to act as a co~ at the junction. For eY~mrle, A.K Covington et al. (Anal. Chim. Acta, 1985, 169, pp. 221-229) describe a open junction where the junction solution is moved from a KCI r~3~. ~oir via a syringe. In this prior art system, the liquid junction is established with each sample, but be~use this type of system leads to cross~ on of the liquids upon use, the junction solution must be discarded along with the sample thus leading to il~Cl~d waste.
Other prior art open free-diffusion liquid junction reference electrodes are gravity fed and thus require specific orientations and geometries to provide good 3 ~

~ 2 1 84954 ~
, ~ ~

reproducible junctions. (See, e.g, R.E. Dohner et al. Anal. Chem., 1986, 58, pp. 2585-2589, T.R. Harbinson et aL, AnaL Chem., 1987, 59, pp. 2450-2456). The orientation and geometries requirements are particularly limiting when allc~ g to adapt such a reference electrode to a mini~hlri~d system.
s Thus, although many different reference electrodes are known in the prior art, there is a need to discover alternative reference electrodes for electrochemical analyzers, particularly reference electrodes that may be easily adapted for use in a mini-integrated type of analyzer.
Sulnlnal~ of the Invention Many ofthe problems associated with prior art reference electrodes have been solved with the discovery of a reference electrode assembly of the present invention.
According to the invention, provided is a reference electrode assembly having a co~ ined-diffusion liquid junction bclwcen a liquid junction solution and a sample solution having se~ "te flow paths, said assembly co~ g (a) a flow cell having lS attached thereto a co~llain~ compri~in~ a region of porous m~teri~l p~rm~ble to water and salts; (b) a remote reservoir for holding said liquid junction solution; (c) a means for moving said liquid junction solution from said les~,l./vil to said co~lld.. l~
and (d) a l~fc,e Ace contact region. Also provided is a mf~,thr~l for providing a ,ef~ ce for a potentiometric sensor analyzer, the method co~ moving a 20 sample solution over at least one in~ ~tor electrode, moving said sample solution t;o a ~,fcl~llcc electrode flow cell to forrn a co~1,d~ed-diffusion liquid junction bc~wcc~
said sample solution and a liquid junction solution over a porous CO~ ,eh.
said liquid junction solution comrri~f,s a non-saluldled e ~ r~ ~,AAl salt and is stored in a remote re~l~o,~ wherein said solution is ~uAulped to said co~ per - t~t 2 1 84954 ~

sample cycle and said liquid junction is electrically connected with sensing equipment by a reference contact region; and measuring an electric potential developed between said liquid junction solution and said indicator electrode.
The inventive reference electrode assembly is particularly useful because it is s not orientation and gravity specific. Also, the assembly may be designed as a multiple use reference electrode for a mini-integrated electrochemical analyzer having planar ~ ed indicator electrodes because sufficient liquid junction solution is present for multiple uses without presenting a space problem because the extra liquid junction solution is stored in the reservoir that is remote from where the co~ ed-10 diffusion liquid junction is created. Other advantages of plefclled embodiments aredescribed herein.
Brief Description of the Drawin~s As described in more detail in the Examples section herein, FIG. 1 is an embodiment of a lere,ence electrode assembly. FIG. 2 shows a top view of a 5 lerel~ nce electrode.
Descli~tion of the ~1~ Çelled Fmbodiments Accoldi~g to the invention, tbe reference electrode has a flow cell where tbe liquid junction solution meets tbe sample solution at a junction co~ ~1 by a region of porous mAtrri~ efll le to water and salts. The porous mAtoriAl generally 20 hinders passage of large molecules (sucb as protein) and bulk liquid ~ Gl l. Such porous Il~Atl ~- ;AiC have been used e~ lc~iv~ly in the art as the "c~ A;-~I" in Coll il~ rl1~iffilcil~n liquid junction ~f~le~ce electrodes and thus are easily ceo~ Able by those skilled in the art and are widely co.. - cially available.
les of such mAt~riAlc are porous plugs, frits andtor mrmbrAn~s~ When selected S ~

;~ 21 84954 ~

as the constraint m~t~ri~l, porous membranes may be fashioned from such m~ttori~1 as cellophane, cellulose acetate, partially nitrated cellulose polycarbonate, combinations thereof, and so on. Particularly preferred, because of ease of use, cost and availability, is a cellophane membrane.
s The flow cell may be fashioned from any suitable m~tPri~1 conducive to the overall design of the analyzer such that the flow cell m~teri~1 is capable of ~tt~ hmPnt to the constraint m~tPri~1, as well recognized by those skilled in the art. If the reference contact region is configured as a part of the flow cell, then the m~teri~1 selected should be non-conductive. If the reference contact region of the reference lo electrode assembly is not a portion of the flow cell, then the m~tPri~l selected may be conductive, if desired.
~tt~rhing the col xll~un~ region of porous m~teri~1 to the flow cell where the junction is formed may be accol,lplished by any number of methods within the skill of one ac~ nled with the art, including for example, bonding or ~ hing with welding, adhesives, mechanical conlplession and the like. The ~tt~mPnt should besecure enough to ~ "1;~l1y ~ /enl regions of dead volume in the jlm- tion solution. Preferably a hPrmetic seal is formed bGlweell the flow cell and the col.x~
m~tP~ri~l The area of the porous m~te,ri~l cA~osed to the sample and/or the junction solution may vary, s~ ni.~ from the entire flow cell to only a small portion of the flow cell, ~le~ upon the specifications of the analyzer design, on so on. The - flow cell may optionally be ~3P*i~P~ such that the porous m~tPri~l covers a distinct cl~ber region of the flow cell, where the co~lldi,ll covers the çh~m~r region and the liquid junction is formed over the chamber region.

' ~ 21 84954 ~

The remote reservoir aspect of the invention functions as a storage unit for excess junction solution. According to the invention, the reservo* is Pxtern~l to the co~ ail,ed-diffusion region where the liquid junction is formed. The external nature - of the reservoir allows for a multiple use reference electrode without requir*ng a buLky s storage unit for the junction solution at the liquid junction itself. The reservoir may be designed in various sizes and shapes, depending upon the protocol of the overall analyzer system and the desired lifet*me of the reference electrode assembly. For example, the l'eSel ~/Oil could be formed by holding the junction solution in the typical tubing (or tubing with a bladder) leading to the region of porous m~t~ l where the 10 liquid junction is formed. Alternatively, the reservoir may be recognized as a separate col~ ler connected by tubing or other such means to the constrained-diffusion liquid junction. Appropriate separate structures that may be used ~ the reservoir include bottles or tanks and so on.
The liquid junction solution comrri~P,s an aqueous solution of a salt having 15 equivalent cationic and anionic co~ ct~nr~s, ~ are well known in the art and include e~luill~u~rel~;lll salts, such ~ KCl, KNO3 and equivalents thereo~ Non-e~luill~r~elll salts, such ~ NaCl, NaBr, KBr, NaNO3 equivalents thereof and so on may also be used, huv~ v~, these lead to h,~lca3ed liquid junction potP ,I;~lc f~ d are e~luil~ r~elll salts (KCl, KNO3, and ~ lulcs thereof). Because of 20 cost and availability KCl is the most pl~,f~,.l~ salt. Other items may optionally be present in the liquid junction solution, inrl~ ing, for eY~n~rlc co.-~p; ~;ble ~ rpnt~, or ions.
The use of silver ions in the liquid jlmction solution is useful in the embo~ where the lef~.lce contact region is a silver based m~tPris~

- ~, 2 1 84954 "
Preferably, when the electrical contact region does not have a barrier membrane and the salt solution (preferably KCl) is non-saturated, then the junction solution further comprises Ag+ ions, the ions preferably present in an amount sufficient to establish stable potentials at the silver based reference contact region but low enough so as to s not induce fouling at the porous m~ten~l constraint. It is known from the prior art that the concentration of Ag+ ions present at saturation is dependent on the co-lce,llldlion of the electrolyte salt present in the junction solution (e.g., see Forbes, G.S., Journ~l of the American Chemical Society, ~, pp. 1937~6 (1911), hereby incorporated by lef~ ce). Preferably, the Ag+ ions are included in the junction solution to the point lo of saturation when the KCl solution is used at a concentration of less than about 2 M.
Preferably when the KCl is present in a concentration of greater than about 2 M to saturation (4.2 M at room lell~cldlure or 7.1 M at 100~C), then the Ag+ ions are provided in a concentration below saturation from about 0.01 mM to about 1 mM, most preferably 0.6 mM. The Ag+ ions may be provided to the salt solution or 5 formed in conjunction with the ~le~alalion of the junction solution by any number of f~mili~r methods. For eY~mplç, the junction solution may be p~ ~l from a dilution of a saturated KCl/s~..n~e~l AgCl solution; ~ i*5~n of solid KCl to a 2 M
KCI/:~tu~ d AgCl solution; dissolution of ap~opl;ate qll~ntitipAs of KCl and AgNO3 in water, and so on.
Any desired eqn~ ~l~alion of the e~ r~ salt in the junction sol~ltion may be utili7~1 Although s~ led solutions of the salt may be used, one of the a~l~,~ti ges of the illv~lltiVt; assembly is that the salt may be used in a c~ n~Pntration below saturation. In a plef~l~d embodiment, a non-s~t~r~ted solution of the salt is employed. Pl~ f~lably KCl is selected as the salt and used in an arnount r~n~ from 2 1 8 9 ~
-about 0.5 M to about 7.1 M, more preferably l M to 4.5 M, and most preferably about 3.5 M. One of the advantages in using a non-saturated solution is particularly evident - when a Ag/AgCl material is used as the reference contact region because with the non-saluldled KCl solution, the Ag+ concentration present at saturation is lowered 5 (thus ~csicting in minimi7ing AgCl dissolution) The means for moving the liquid junction solution from the reservoir to the col~llaiIll region may be selected from various known devices, taking into effect the overall design of the analyzer. Included in the definition of devices that may be selected for the means for actively moving the liquid are devices such as pumps, ~ylhlges~ and the sort. For automated systems, a peristaltic pump, reciprocating syringe or other such device is particularly useful.
The design of the analyzer may be such that the device used for movement of the junction solution may also be used for moving other liquid(s) in the analyzer, such as, for example, the sample solution, calibration solution, wash solution, combinations 5 thereof, and so on. ~lt~rn~tively, a device specific to movement of the liquid junction solution alone may be used. The flow rate and volume of the liquid junction solution may be adjusted to the specifications of the analyzer to provide a conr,~n~tion of electrolyte salt at the liquid junction that res~mhles that of the salt solution of the l~wl ~oih, as recog~i~abl~ to those skilled in the art.
The ,~,f~,~ce contact region may be placed a~y~ll~.e within the analyzer as long as it may fim~fion so that electrical contact is rnade ~lwee~ the sensing e~ and the liquid junction solution. Suitable ..,i~t~ for the l~f~e~ce contact region are m~t~ri~lc including con~ ctive metals, alloys thereof, and composites thereof having ~cc~ble conductive ~ro~Gllies, as are easily reco~i7~d 2184954 ~

by those skilled in the art (i.e. materials that are capable of providing an electrochemically reversible redox electrode couple). Particularly suitable are silver - based materials and calomel (Hg/Hg2CI2). More preferably silver based materials are used (e.g., Ag/Ag+and Ag/AgCl) . Although barrier membranes covering all or a 5 portion of the electrical contact regions may be used, for ease of use, preferably no such barrier membrane is present in the reference electrode.
For ease of use, the l~fe,~llce contact region may be designed into multiple conductive regions that are connected together to f~cilit~te electrical contact b~lw~;en the sensing equipment and the liquid junction. For example, two regions may be lO used. In this design, the first region is where the contact is made with the liquid junction solution, with the first region prepared from m~trri~l~ that are capable of providing an electrochemically reversible redox electrode couple. The second region is directly comle~;ted to the first region but does not necess~. ;ly touch the liquid junction and may be prepared using any suitable electrically conductive m~trri~l~ (i.e.
5 ~ g beyond those m~teri~l~ capable of providing an electrochemically reversible redox electrode couple). In this design, the funcffon of the second region is to f~ilit~te making electrical contact to the sensing eqllirm~nt Illu~ ive of this set up would be a first region col~ ;sing a con-luctive wire co~ ~t~d to the liquid jlmctirm solution and a second region that is a "pad" embe~lded or screen printed onto the non-20 col~lu~,1iv~; flow cell. In this embo-lim.ont the wire would be set into the pad in a ~l~ndicular fashion. The electrical contact(s) from the sensing e~ would touch the second region pad, thus providing indirect contact b~tw~ll the sensing e,l..;r~ rnt and the liquid junction solution.

4~ 2 1 84954 ~

The reference electrode assembly may be used in conjunction with one or more potentiometric indicator working eleckode(s) whose response depends upon analyte concentration. Ion-selective electrodes based on solvent polymeric membranes are examples of such indicator electrodes. Examples of analytes that may s be measured include, for example, chloride, potassium, calcium, sodium, pH, bicarbonate, magnesium, and so on. The reference electrode and t~he indicator electrode con~lilule individual galvanic half-cells which together comprise an electrochemical cell which allow for potentiometric ~ re analysis. The l~rGIGllce electrode assembly is capable of providing a steady and stable potential 10 sufficient for potentiometric analysis over the clinically relevant range of ionic strengths, protein concentrations, and h.om~t~rit levels. Optionally, the reference electrode assembly may be packaged together with mini-integrated working electrodes stationed on a sensor module such that the entire configuration is a single disposable unit.
1S It is to be understood that various modifications to the invention will be a~ ~ll to and can readily be made by those skilled in the art, given the disclosure herein, without departing from the scope and m~t~ri~l~ ofthis invention. It is noted ~at the following examples given herein are int~n~1ed to illll~te and not to limit the invention thereto.
20 - Examples - Many configurations ofthe reference electrode ~ n hly may be de~
- FIGs. 1-2 illustrate a configuration of a referellce electrode assembly inco.~l~l~d into a mini-illleglaled ele~,~ocl~lllical analyzer 1 where the reference electrode has a lifetime of at least about one month with typical clinical usage.

2 1 8 9 5 ~3 More particularly, in FIG. 1 the schematic view illustrates a reference electrode 5 positioned behind an array of a plurality of working electrodes 6. The junction solution is pumped from a remote ext~m~l reservoir l0 located apart from the liquid junction region of the reference electrode 5. The orientation of the reservoir 10 5 is not gravity specific, and thus the reservoir may be placed anywhere that is convenient to the other items of the analy~r. From the sample entry 12 portion of the sensor array, the analyte co~ g sample is pumped through the various working electrodes to a sample exit 14. The sample solution flow path and liquid junction solution flow path are s~l~; paths. A comunon pump lS is used for 10 sample solution movement and junction solution movement. The junction solution is moved from the remote reservoir l0 to the site of the reference electrode 5 each time a sample is run. The movement of the junction solution may be continuous but more efe.dbly occurs only at such time as when a sample (or reference liquid such as a calibrator or quality control m~t~.ri~l) iS present. Once used, the junction solution may 15 be discarded as waste or recycled and pumped back to the r~s~/oil 10. Preferably the solution is recycled, thus r.l~ Ie~ g the lirelilllc of the 1~ relel~ce electrode and also ;..g waste. In a particularly pler~ll~ configuration, the liquid junction solution is re-circulated and pumped with the intro~l17ction of each analyte sample and b~..-- -s st~nt after the testing of the sample is comrlete~l Pl~ f~.dbly, only a 20 small volume of the lere~ ce electrolyte solution is moved to the co~ region of the i~f~r~.lce electrode S and optionally recycled back to the t-Yt~o.rn~l LeS~./oil 10 per cycle, thus allowing for a small front-end lef~,e,lce electrode assembly particularly useful in a mini-integrated analyzer.

' 1 2 1 8 4 9 5 4 ~

As shown in FIGs. 1 and 2, the liquid junction solution may be pumped from the reservoir 10 to an inlet tube 16 over the region of porous m~tPri~l forming the COII~ ai~l~ 18. The porous material (preferably a permeable membrane) is ~tt~hed to the flow cell by f~ctçning a gasket over the material whereby a hermetic seal between 5 the porous material and the flow cell is formed. The area constrained by the porous m~teri~l is where the sample and the junction solution int~rf~re in the flow cell thus forming the liquid junction. The flow cell has a defined elliptical shaped chamber, where the shape of the chamber helps provide an effective washout of the entire chamber and also .,.i~ s bubble trapping. Junction solution depletion occurs at a lO rapid rate once the junction solution is held st~gn~nt in the flow cell. Design of the surface areas and volume of the elliptical chamber relative to the sample volume and hold time may be manipulated to provide the desired p~r~,- ...~nce Preferably when such an elliptical shaped chamber is used, the col~ region is ~tt~hed thereto. In a yl~f~ d embodiment, the elliptical chamber has a volume of from about 3.5 to 5 about 17 ~lL, most preferably about 3.5 IlL. The electrical contact region has two parts. The first region 11 is a Ag or Ag/AgCl wire that is imbedded in a second contact region 25 that is attached to the non-con~ ctive flow cell. The second region 25 has an ~ A~osed portion in the flow cell through which the electrical contact is made~
with the sensing e ~ The sample is removed from the sensor array through the 20 sample exit 14 as waste while the jlm~ n sol~ltion is recycled back to the le3~ Oil 10 via the exit tube 17.
In the embodiment shown in FIGs. 1 and 2, the reference electrode most ~ r~l~bly employs a non-s~tulaled KCl liquid junction solution stored in a lese. VOil having a volume of from about 1 rnL to about 50 mL (most plef~.~bly 8mL), a ~--~ 21 84954 ~3 membrane porous constraint region, and a silver based reference contact region without a barrier type of membrane. Advantages of this embodiment include a - multiple-use capability, with minimum fouling of the porous m~t~ 1 region over the use lifetime. The assembly may be stored as a dry reference electrode assembly. The s KCl junction solution may be released form the reservoir which then easily wets the flow cell upon first use, where bubble formation at the membrane region (which typically accol~a~ies a first use) is reduced. Also, in this pler~lled embodiment, the junchon solution is re-circulated and thereby greatly minimi7~s the reservoir volume necess~y for multiple use and also reduces the amount of waste generated as lo co",p~d to systems with one-use only solutions. This reference electrode also may be stored at sub-ambient tell~eldlu,es with a reduced possibility of salt cryst~11i7~tion while providing reproducible results having an acceptable accuracy with multiple testing capability. In this embodiment there is exhibited an adequate ionic strength independence at relatively low junction solution concentration, as low as about 0.5 M
5 KCI. This is ul~e~e~ ted based on the Henderson equation for the liquid junction potential of two freely diffusing liquids.
Example 1 R f~l~"ce flow cells were either ~ hil)ed from rigid acrylic or injection molded using ABS plastic. For each flow cell constructed, the CQ~ m~te~i~l was 20 a ion and water p~ ...f~ble free-st~n~ling 0.001" thick cellophane (1--co~ted ege~ .dled cellulose) film membrane obtained from Flexel, Inc., Atlanta, GA. The film was placed over an elliptical shaped chamber of the flow cell (the chamber having a volume of appro,~ 1y 3.5 ,uL). The film was h~.~m.ofi~lly sealed to the cl~ region of the flow cell with co",~s~ion from a gasket on top of the flow r~ 2184954 ~

injection sensor module assembly. Sample solution and liquid junction solution flowed past each other on opposites sides of the surface of the cellophane film membrane at a flow rates of from about S to about 100 11L/sec. A common peristaltic pump w~ used to simultaneously move both solutions. Electrical contact 5 was established via an electrochemically plated 0.012" Ag/AgCl or Ag wire located either within the remote reservoir solution or in the base of the flow cell imbedded in screen printed epoxy silver in a perpendicular fashion to extend into the inlet tube of the flow cell. The sensing equipment used was a HP 3457A digital multimeter. The total volume of the solution in the junction reservoir ranged from approx. 8 mL to 0 approx. 15 mL. The junction solution was moved from the remote reservoir to the flow cell and past one surface of the cellophane membrane with every sample solution. The junction solution was then either re-circulated back to the reservoir or sent to waste after the measurement cycle. Electrode me~u,~ lents were taken when both the sample and liquid junction solutions were stationary.
Various junction solutions were tested. The aqueous based solutions teste were (a) 1.5 M of KCl s~ led with AgCl (approx. 0.25 mM); (b) 2.0 M of KCl saturated with AgCl (approx. 0.6 mM); (c) 3.5 M of KCl with 0.6 mM of AgCl; and (d) 4.0 M of KCl with 0.6 mM of KCl, where some of the solutions further cr....l.. ;~ed about 0.05 g/L of BRIG'9 700 (...~....I~.j(...~;d by ICI Sl~rfi~cPnt~, Wilimin~n, Del.).
The wet up of a fully dry system was established to co.. ~,.cially de~ilable specifications in less than five ~ es Ionic ~ ~ depon-lenre over a çlinir~lly significant range of 0.120 mM to 0.200 mM was evaluated. Reference l,ot~lllial changes of less than 0.5 mV were measured for all solutions tested over a time period a~ g approx. 1 month. This reference electrode was used toge~er with se~

~ 2 1 84954 ~

planar ion-selective electrodes sensitive for pH, potassium, ionized calcium, and sodium for the analysis of these analytes.
Example 2 Reference electrodes were prepared as described in Example 1 with the s following exceptions.
The placement of the reference contact region (a Ag wire) was varied. A three dimensional sodium ISE working electrode (200 Series obtained from Ciba Corning Diagnostics Corp., Medfield, MA) was tested with three reference electrodes using a junction solution of 2M KCI/saluldted with AgCl. The reference electrode used as the o control was a 200 Series Corning Diagnostics Corp three-dimensional type of ,ef~cnce electrode. The calibration reagents were that used on the Ciba Corning Di~gnostics Corp. 644 Instrument.
In Set A, the reference electrodes tested had a Ag wire located in the base inside the elliptical chamber. A reference electrode ~r~aled with a Ag wire in the b~e of the flow cell was tested over a period of 35 ~ es. The average within sample drift was -0.047 mV/sec. A reference electrode with a Ag wire in the remote lCs~/oi, was tested under the same conditions and was found to have an average within sample drift of -O.OOl mV/sec. The control 200 Series ~efe,ellce electrode had an average within sample drift of-0.0004 mV/sec.
In Set B, the reference electrodes tested had a Ag wire located in a base outside the elliptical chamber. A reference electrode p,~,d with a Ag wire in the base outside the elliptical chamber of the flow cell was tested over a period of 35 .~-;....~es. The average within sample drift was O.OOl mV/sec. A reference electrode with a Ag wire in the remote reservoir was tested under the same conditions and was ~ 2~84954 ~

found to have an average within sample drift of 0.0004 mV/sec. The control 200 Series reference electrode had an average within sample drift of 0.001 mV/sec.
Example 3 Reference electrodes were pre~ar~,d as described in Example 1 with the s exception that the elliptical chamber had a larger volume (16.8 111) as colllpaled with the elliptical chamber of Example 1 (which had a volume of 3.5 1ll). A three dhllensional sodium ISE working electrode (as described in Ex. 2) was tested with three reference electrodes using a junction solution of 2M KCl/saturated with AgCI.
The reference electrode used as the control was a 200 Series Corning Diagnostics lo Corp three-dimensional type of reference electrode. The calibration reagents were that used on the Ciba Corning Diagnostics Corp. 644 Instrument. The reference electrodes tested had a Ag wir~ located in a base inside the elliptical chamber. The electrodes were tested over a period of 35 ...i~ es. The average within the sample drift was -0.012 mV/sec. When repeated the average within the sample drift was -0.028 15 mV/sec. The control 200 Series reference electrode had an average within sample drift of 0.001 mV/sec.

17,

Claims

1. A reference electrode assembly having a constrained-diffusion liquid junction between a liquid junction solution and a sample solution having separate flow paths, said assembly comprising:
(a) a flow cell having attached thereto a constraint comprising a region of porous material permeable to water and salts;
(b) a remote reservoir for holding said liquid junction solution;
(c) a means for moving said liquid junction solution from said reservoir to said constraint; and (d) a reference contact region.

2. A reference electrode assembly according to claim 1 liquid junction solution comprises an equitransferent salt solution.

3. A reference electrode assembly according to claim 2 wherein said salt solution is present in a non-saturated concentration.

4. A reference electrode assembly according to claim 3 wherein said constraint is a membrane.

5. A reference electrode assembly according to claim 4 wherein said membrane is cellophane attached to said flow cell with a hermetic seal.

6. A reference electrode assembly according to claim 5 wherein said equitransferent salt is KCl present in a non-saturated concentration.

7. A reference electrode assembly according to claim 1 wherein said junction solution comprises a KCl salt present in a non-saturated concentration.

8. A reference electrode assembly according to claim 7 wherein said junction solution further comprises Ag+ ions and said reference contact region is a silver based conductive material.

9. A reference electrode assembly according to claim 8 wherein said Ag+
ions in said junction solution are present in an amount of about 0.01 mM to 1 mM and KCl is present in an amount ranging from about 1 M to saturation.

10. A reference electrode assembly according to claim 8 wherein said Ag+
ions in said junction solution are present in an amount of about 0.6 mM and said KCl is present in an amount ranging from about 2 M to about 4 M.

11. A reference electrode assembly according to claim 10 wherein said reference material consists essentially of bare Ag or Ag/AgCl; said means for moving said junction solution is a pump; and said junction solution comprises an equitransferent salt present in a non-saturated concentration and Ag+ ions.

12. A reference electrode assembly according to claim 1 wherein said junction solution is recycled back to said reservoir after functioning at said liquid junction per sample measurement cycle.

13. A reference electrode assembly according to claim 12 wherein said volume of the remote reservoir is from about 1 mL to about 50 mL.

14. A reference electrode assembly according to claim 13 wherein said volume of the remote reservoir is about 8 mL with a one month use life.

15. A reference electrode assembly having a constrained-diffusion liquid junction between a liquid junction solution and a sample solution having separate flow paths, said assembly comprising:

(a) a flow cell having a chamber region wherein a region of porous membrane constraint permeable to water and salts is attached to said chamber region of said flow cell;
(b) a remote reservoir for holding said liquid junction solution comprising an equitransferent salt and Ag+ ions, wherein said reservoir is a separate container connected by tubing to said region of porous membrane constraint;
(c) a pump for moving said liquid junction solution from said reservoir through said tubing to said constraint; and (d) a reference contact region comprising a silver based material.

16 A method of providing a reference for a system for potentiometric quantitative analysis comprising:
(a) moving a sample solution over at least one indicator electrode;
(b) moving said sample solution to a reference electrode flow cell to form a constrained-diffusion liquid junction between said sample solution and a liquid junction solution over a porous constraint wherein said liquid junction solution comprises a non-saturated equitransferent salt and is stored in a remote reservoir where said solution is pumped to said constraint per sample cycle and said liquid junction solution is electrically connected with sensing equipment by a reference contact region; and (c) measuring an electric potential developed between said reference contact region and said indicator electrode.

17. A method according to claim 16 wherein said liquid junction solution is recycled after (b) to said remote reservoir.

18 A method according to claim 17 wherein said liquid junction solution is stagnant until such time as step (a) is performed.

19. A method according to claim 16 wherein said reference contact region is a silver based conductive material and said junction solution comprises (a) KCl present in an amount of about 3.5 M and (b) Ag+ ions present in an amount of about 0.6mM.

20. A method according to claim 19 wherein said indicator electrode is a planar miniaturized electrode.

21