CA1138043A - Electrochemical testing system - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
An electrochemical measuring system which effects, measures and sorts charge transfer reactions of selected substances in a sample solution is provided. The system includes a novel electrode which is adapted to measure simultaneously on at least two elec-trodes at least two electrolytic potentials with reference to a reference potential. In a preferred form the electrode comprises a hollow, cylindrical body formed of an electrically insulating material. The electrode is open at least at one end and has a gen-erally smooth cylindrical inner surface. A plurality of electrically discrete active electrode segments are mounted on the inner sur-face of the electrode with their active surfaces substantially flush with the generally smooth cylindrical inner surface. Com-pleting the electrochemical measuring system are a stirring means for creating a relatively high degree of mixing adjacent the electrode active surfaces, means for charging liquid samples to the cell, and means connecting at least two of the electrically discrete active electrode segments to different electrical potentials. In use a sample to be tested is charged to the cell, and stirring is commenced. One of the electrically discrete active electrode segments is held at a potential at which a selected substance of interest and also one or more interferring substances responds, while another of the electrode segments is held at a potential at which only the interferring substances respond. The presence of a substance of interest can be determined by subtracting the signals from the one and another electrode segments, and its quantity determined by integrating the signal difference.

Description

~ 3~0~3 Various electrochemlcal systems are known in the art Eor detectin~ the presence of and/or measuring the concentration of various substances of interest in sample solutions suspected of containing the selected substances, and find utility in a variety of environmental, medical and industrial applications.
Generally, such systems are employed in analyzing for metallic ions of interest, although systems also exist for the detection of non-me~als such as cyanide ion, sulfur dioxide and halogen, and for certain organic materials.
One type of prior art electrochemical analysis employs gravimetric methods in-which a deposit formed by electrical action is weighed on an analytical balance. Gravimetric methods are prone to weighing errors, require a skilled technician, and are relatively time consuming and insensitive.
Another type of prior art electrochemical analysis employs ion-selective electrodes. A number of ion-selective electrodes have been devised for testing for a variety of ions of interest and are considered to be reliable and relatively easy to use.
However, a number of substances of interest in the environmental, industrial and medical fields cannot be measured with ion-selective electrodes. Moreover, ion-selective electrodes respond logarithmically and thus generally are not sufficiently sensitive for measuring concentrations below about l0 5 to l0 6 molar. -' Polarographic analysis based on current voltage curves obtained with han~ing drop mercury electrodes offers an advantage over ion-selective electrodes of sensitivity in dilute solutions.
A feature and requlrement of classlc hanying clrop mercury polarographic electrolysis cells is the dropping mercury elec-trodel i.eO, mercury droplets being discharged periodically .
into a solution from a fine bore capillary under a driving head of mercury. However, this very feature, which has permitted the initiation of extremely useful polarographic methods in research work, mitigates against a more general use of classic polarogra-phic electrolysis cells as common analytical systems, and in particular as tools for monitoring and controlling industrial process streams or for field use testing in mediral and environmental applications. Moreover, the characteristic periodic grow~th and fall of the~mercury droplets cause oscillations in the current-voltage curves ob-tained using such cells and thus prevent the establishment of standard curves. Other problems of hanging drop mercury electrodes which have essentially limited cells employing same to laboratory and experimental use include condensor current build-up whenever a new mercury droplet is being formed at the capillary, and limited surface area of the droplets which limits sensitivity of the electrode. In addition, .
formation of the tiny mercury droplets is a delicate process .
which may b~ affected by a number of incidental factors, including mechanical vibration, slant of capillary, and pulsation of test solution into the capillary inlet between drops. In this connec-,.
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tion it should be noted -that the reproducibility of droplets with reyard to their drop line and mass of mercury per drop must be practically perfect at all times to permit proper evaluation of the polarogram.
Still another type of prior art electrochemical measuring system is a technique called coulometric s-tripplng voltamme-try.
Coulometric stripping voltammetry is a two-step process comprising electrodepositing the electroactive material of interest on or in an indicating or working electrode and then electrodissolving or stripping the deposited material back into solut.ion. In anodic stripping voltammetry the material to be measured is plated onto an electrode by applying a negative potential over an extended time period, and then stripping the material off the electrode over a relatively short period by sweep1ng to a positive potential~
The order or potential at which the elements of the material are stripped off ~he electrode provides a qualitative analysis of the material, and the quantity of the current provides a quantitative analysis. Anodic stripping voltammetry offers the advantages of enhanced sensitivity, resolution, and reproducibility compared to classical polar- I
ographic analysis obtained using hanging drop mercury electrodes.
By way of example, thin-film mercury/graphite composite elect~odes have been employed in anodic stripping voltammetry sy~tems for ana lyzing for metals at the sub-nanogram level. See, for example, the reported work of Wayne R. Matson, Reginald M. Griffin~
ard George B. Sohreiber in "Rapid Yub-Nanogram Simultaneou~

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~nalysis of Zn, Cd, Pb, Cu, Bi and Ti", Trace Substances in 1 H~alth, Unive.rsity of Missouri, Dr. D. Hemphill, Ed; pp. 396-406, (1971). While electrochemically analyzing solutions employing composite mercury/graphite electrodes by anodic stripping voltammetry, e.g. as taught by Matson et al, supra, may provide sub-nanogram sensitivity, the ability to rapidly and reliably differentiate and measure selected substances at the picogram level is not generally possible using existing electro-chemical measuring techniques. Also, many metals interact with the electrode to form an alloy or analgam. Thus, anodic and cathodic stripping vol.~ammetry are limited to detection of a rela- -.
tively small number o species of metals and non-metals. Obvious-ly, the ability to operate at such low concentrations and on a wider variety of species would have major commercial utility in environmental, medical and industrial applications.
It is thus a primary objec~ of the present lnvention to provide a novel and improved system, i.e. method and apparatus, which overcomes the aforesaid and other problems and limitations Gf the prior art.
Another primary object is to provide a novel and improved method and apparatus for electrochemically analyzing a sample in oxder to qualitatively and/or quantitatively determine the pre- I .
sence of selected substances in the sample.
Another object of the present invention is to provide an electrochemical measuring system of the aforesaid type which is capable of rapidly and reliably operating at the picogram level of sensitivity.
A more specific object is to provide a novel and improved electrode for use ln electrochemical systems.
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ESA~117 CIP -4- 1 .

l:~L3~1043 In order to ef~ect the Eoregoing and other objects there is provided an electrochemical measuring system which effects, measures and sor-ts charge transfer reactions oE selected substances in a sample solution. The system includes a novel electrode which is adapted to measure simultaneously on at least two elec-trodes at two electrolytic potentials with reference to a reference potential. In a preferred form the electrode comprises a hollaw, cylindrical body formed of an electrically insulating material.
The electrode is open at least at one end and has a generally smooth cylindrical inner surface. A plurality of electrically discrete active electrode segments are mounted on the inner surface of the electrode with their active surfaces substantially flush with the generally smooth cylindrical inner surface. Completing the electrochemical measuring system are a stirring means for creating a relatively high degree of mixing adjacent the electrode active surfaces, means for charging liquid samples to the cell, and means connecting at least two of the elec-trically discrete active electrode segments to different electrical potentials. In use a sample to be tested is charged to the cell, and stirring is commenced~ One of the elecirically discrete active electrode segments is held at a potential at which a selected substanc~ of interest and also , one or more interferring substan~es responds, while another of the electrode segments is held at a potential at which only the l~

l~L;:~8~343 interferring substances respond. The presence of a substance of interest can be determ:inecl hy subtracting the signals from the one and another electrode segments, and its quantity determined by integrating the signal difference.
Yet other objects of the invention will in part appear obvious and will in part appear hereinafter. The invention accordingly comprises the apparatus possessing the construction, combination of elements, and arrangement of parts, and the process comprising the several steps and the relation of one or more o such steps with respect to each of the others, all of which are exemplified in the following detailed description, and the scope of the application as will be indicated in the claims~
For a fuller understanding of the nature and'objects of the present invention, reference should be had to the ollowing de~'àiled description ~aken in connection with the accompanying drawings wherein: j Fig. l is a front view in pe~spective of a preferred form electrochemical measuring apparatus according to the invention; I' Fig. 2 is a front view, partially in seGtion, ol a preferrea form of electrolytic cell of the apparatus of Fig. 1, Fig. 3 is an end view, in cross-section of the sample solution stirring member of apparatus of Fig. l; ~ ¦
Fig. 4 is a fragmentary plan view, in cross-section o a pre-,- -,~
ferred form of sensing electrode element of the apparatus of Fig. l;

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113~30~3 Fig. 5 is a diacJranunatic view of a flow detec-tor element of the apparatus of Fig. l;
FicJ. 6 is a block diayram of the electrical and pneumatic controls and functions of the apparatus of Fig. l;
Fig. 7 is a plot showing the current in microamperes, ~A, verses the potential in volts verses a standard silver/silver halide reference electrode, obtained in accordance with the present inven~ion; and Figs. 8 and 9 are ~ront views, in perspective, o~ alternative iorms of electrolytic cells in accordance with the present invention~
The present inven~ion is based on measurements oE
electrochemical reactions of selected substances in solution under controlled potential conditions. As is well known in the art, when electroactive substances are dissolved in a solvent to form a reagent or electrolyte~ and an electrical current passed through the electrolyte between an anode and a cathode disposed therein, positive ions will be attracted to the negatively charged cathode where their charge will be neutralized, while negative ions -will move towards and be discharged at the anode. The electrical potentials at which such electrochemical reactions occur will vary depending upon the particular substances involved. By way of example, consider an aqueous solution which contains both iron and copper ions. Iron normally exhibits a valance of two or three, while copper normally exhibits a valance of one or two. The electrical potential at which ferric ions (Fe ) in solution may be reduced to ferrous ions ~Fe ) is a constant at a given tempera~ure. Likewise, the electrical potential at which cupric ions ~Cu ) in solution may be reduced to cuprous ES~-117 CIP ~7 . . ~

ions (Cu+l~ is also a constant at a given temperature, and is different from the electrical potential at which the reduc-tion of ~erric ions to ferrous ions occurs. (The electrical potential at which such reac-tions occur are approximately described by tables of s-tandard or formal po~entials).
The absolute value of the electrical potential of ions of solu-tion is indeterminate. However, electrochemical reactions for a particular species are described in terms of a potential versus a standard reference couple such as H2/H . The magnitude of the potential is a measure of the potential that has to be applied versus a standard reference electrode to force charge transfer to occur. The electrical potential at which such reactions will occur is referred to ~s the "Charge tran,fe~ potentia1".

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Assigning an arbitrary value of zero to hydrogen, the potential E of an electrochemical reaction may thus be written according to the following reaction:
E = Eo - 0~05915 loglo (~P) (l) where n is the number of Faradays, Ap and AR are activities of the product reactants, and x and y are corresponding co-efflcients of the electrochemical reactions~ Thus, the potential Eo is the standard potential related to the par-ticular reaction.
E is a potential applied to drive the reaction either to reactants or products according to the equilibrium condition described by equation (l). Under conditions where the ~ applied is large enough to drivè the reaction to virtual completion at equilibrium, the current derived will be proportional to the concentration of the reactant in the solution. However, background ~oise prevents direc~ measurement of most sample solutions and in the case of very dilute solutions may prevent direct measurement in many instances. (As used herein the term "background noise" is intended to refer both to major interference factors such as the presence in the solution of other electroactive materia1s which, by virtue oE their e1ectrica1 activity in the 11 3~ 4;~

solution, respond to the same electric~l potential as the ion of interest, and also major non-Faradaic in~erference factors such as capacitance signals of the electrode in the solution due to the existence of a boundary layer of still solu-tion adjacent the active surfaces of the electrode, bulk solution signals, in-herent Faradaic signals, elec-trode settling signals and the like). ..
A feature and advantage of the presen-t invention resides in the elimination of and/or cancellation of background noise through a combination of electrochemical manipulations and electrode geometry.
Further understanding of the features and advantages of the present invention will be had from the following detailed description of one preferred embodiment of the invention which illustrates an electrochemical testing system for measuring .
the iron content in serum or blood. It will be understood .
however, that the system of the present invention may be advantageously employed for detecting the presence of and measuring the concentration o~ various other substances of interest in sample solution.
Referring to Fig~ 1, there is illustrated an electrochemical measuring apparatus indicated generally at 10 including a~base 11. Mounted Qn base 11 by means of upright support 12 is a cabinet 13 whose ~ront face acts as a control panel 14.
Mounted on the panel are various control means including a :~.38(;~

display panel 16, Eunction buttons including a stand~y button 17, an "autoblank" control button 18, an "au~oblank set"
button 19, and a calibration ]cnob 20. Also positioned on -the control panel 14 is an ofE-on button 22, a flow indicator 23 suitably labelled to show that a prior sample is flushed out and a new test may be started. Also on the control panel are a start button 24, and a "running" indicator 25.
For convenience it is preferred that the controls be combinations of push-buttons and indicating lights 9 and in the actual apparatus such combination buttons and lights are used.
Depending from the bottom of cabinet 13 is a cell assembly 27 indicated in outline and shown in further detail in Fig. 2.
Positioned on base 11 are two containers 28 and 29, suitably connected by plastic tubing or the like to the cell block.
Container 28 receives flushed cell contents at the end of each run and container 23 holds a supply of fresh cell liquid or electrolyte. -Fig. 2 shows the cell assembly of the apparatus o Fig. 1 comprising generally a cell block 33 and a sensing electrode 34 mounted therein. Cell block 33 comprises a suitable mounting piece such as, for example, a plastic block having a screw threads 35 or other mounting means at the upper end. A vertical channel ' or cylindrical hollow 36 runs through the cell block and com-municates with the interior of-sensing electrode 34. Two passagewaysl the first an inlet passage 38 to receive a sample to be tested which may~ for example, be by means of a pipette ~3~ L3 ~not shown~ inserted into channel 38, and the second an outlet passageway 39 for cell liquid. Cell block 33 is formed of a liquid-impervious, rigid, electrically insulati~g, chemically inert material such as unplasticised polyvinyl chloride, poly-tetrafluoroethylene fluorocarbon resins or the like.
The bottom of ~hannel 36 is recessed to receive sensing electrode 34. Sensing electrode 34 is in the form of a hollow cylinder, and the inner surface of the elec-trode and the inner surface of channel 36 are flush and as smooth as possible so as to minimize the material caught therebetween. In actual practice the electrode is permanently mounted in the cell block by suitable means such as, for example, by an epoxy resin or the like, and the inner surface of the joint between the two is machined smooth.
At the bottom of sensing electrode 34 is a seal and ~ , ..
connector device 46 which may, for example, be in the form of a plastic plug molded to the sensing electrode 34 having a screw thread connection 35a for connecting a pipe or hose thereto and having a channel 49 extending therethrough. A continuous passage is thus formed, and electrolyte or other contents of the cell can be flushed out by passing fresh electrolyte or other liquid in through channel 49 and out through outlet 39 in the cell block above the electrode.

1 1.38043 ~ s rnenti.onecl supra an important fea~ure and advantaye o~
the present inventlon is the ability to differen-tiate between electrical signals represen-tative of the charge transfer electrolytic reaction of selected substances of interest, and electrical signals derived from the bulk sample solution, inter-ferring substances and other background noise. This featuxe and ..
advantage is made possible in part by the construction of sensing electrode 34. Sensing electrode 34 comprises a generally cylin-drical body of block epoxy having moun-ted therein a plurality of active electrode segments. The electrode body comprises an electrically .insulating material such as a polymeric material while the active electrode segments comprise a suitable electrode base such as graphite, pyrolytic graphite or platinum, or the active electrode segments may comprise coatings of active electrode matexial such as mercury or gold. In practic~, at least two electrically discrete electrode areas are employed, for example, in the form o~ rings or bands of active electrode surface on the inside of a hollow electrode body. The segments are separated by electrically insulating bands on the inside of the electrode. Such an electrode can be formed by holding segments of active electrode material in the form of rings in desired posi-tion and molding the rings with an electrically insulating such~as an epoxy resin to form a cylinder. Sensing electrode 34 is shown in further detail in Fig. 4. For convenience - , uf illustration sensing electrode 34 has been shown as comprising two active testin~ electrode segments, a counter or power supply-ing electrode segment, and a reference electrode segment as follows:- a first active testing electrode segment 42, a second active testing electrode segment 43, a third counter electrode segment 44, and a reference electrode segment 45. The first, second and third active electrode segments 42, 43 and 44 are ,.
formed of suitable electrode material such as graphite or the like while the reference electrode segment 45 is formed of silver palla- .
dium or the like. Electrode segments 42, 43, 44 and 45 each com- :
prise a cylindrical ring embedded into a cylindrical electrode body :.
41. The electrode segments are spaced apart by'a narrow gap so as ~, to be electrically insulated one ~rom the other, and the electrode segments are mounted so that the active surfaces are substantially flush with the inner surface of electrode body 41 so that the ~
inner su.rface,,of the entire el~ctrode 34 is a smooth as possible. ' Electrical connections (not shown in Fig. 4) are provided to each of the electrode segments and are suitably connected to the apparatus ,, by means of a four wire lead terminating in a four-pronged plug as shown in Fig. 2. Obviously the electrode may comprise additional active electrode segments.
Positioned within electrode 34 is a stirring means 50. .
Stirring means 50 is mounted for rotation within the electrode' body by means of rod 51. Stirring means 50 and rod 51 are formed of electrically insulating and chemically inert materials E52.--117 CIP -14--.

such as molclecl r~sin. The lower end of stirring means 50 is sli~htly wedge-sllaped or cone-shaped, and is generally close fitting within the electrode body. A diagonal groove 52 which is better seen in Fig. 3 r~ns along the surface of the stirring means 50. ~hen rotated in the direction shown by arrow 54, groove 52 creates a high degree of mixing or turbulence closely adjacent to the active surfaces of electrode segments 42, 4~, 44 and 45 so as to minimize the thickness of the boundary layer of still solution adjacent the active surfaces of the electrode segments, while maximizing mass transfer to the electrode surfaces.
In use off-on button 22 is ~irst activated. Ordinarily, the apparatus will be left running in a standby condition overnight and will be turned off if it is to be left idle for a period of a week or more. At the start of each week, or for purposes of abundant caution at the start of each day, the apparatus may be calibrated. It is first operated with the calibration button in operating position to standardize the electronics as will be hereinafter described. A blank sample of reagent is run first. Then the "auto-blank" button l9 is set, holding the calibration. Next a standard sample of known ion concentration is introduced into the cell 27 and the appa-ratus run through a cycle. When it has been properly stand- !
ardized, the calibration knoh 20 is adjusted so that the reading in the d7splay panel 16 corresponds with the known ion quantity in the standard calibration sampleO

~.~13~ 3 A plastic tube or pipe 40 (not shown in E~ig. 2) connects the cell assembly 27 to the apparatus. At a selected point along tube 40 and preferably within cabinet 13 is a flow detector illustrated diagrammatically in Fig. 5. An emitter 55 or other light source is positioned near a window at a point along tube 40. The window may be a transparent insert or the tube itsel may be transparent. Opposite emitter 55, i.e. on the opposite side of tube 40 is a detector 56 posi-tioned adjacent a similar window. When tube 40 is empty or filled with a gas the beam of light 57 from the emitter is quite diffuse. When tube 40 is filled with a liquid such as the cell electrolyte flowing through the tube, the liquid acts as a lens and increases the sharpness of ~ocus of light beam 57.
Detector 56 is adjusted for a threshold such that it can deter-rnine the,,pr,e,sence of liquid in ~ube 40 and the length of time such liquid is present. The signal from detector 56 is employed to indicate that there has been flow of liquid through l- -tube 40 for a sufficient time to accomplish flushing out of cell electrolyte after a single run so as to remove the sample therefrom.
In repetitive runs the cell 27 is repeatedly filled with an electrolyte and the cell stirring apparatus is con,stantly in operation to keep the cell contents uniform and mixed. A
known quantity of a test samplé is then pipetted into a cell 2/.

113~3U~3 The running lndicator 25 lights to show that the -test is in oper-ation. In a preferred embodiment of the present invention the dis-play panel is a digital display which counts to zero and then up to the number of micrograms per lO0 mililiters of serum (~g%~. When the digital display stops counting the test is complete. After a timed waiting period the cell electrolyte containing the sample is flushed into container 28 and a new supply of electrolyte is introduced into the cell from container 29. When the start test indicator 24 lights up again, the apparatus is ready for a next sample.
In Fig. 6 is shown a block diagram of electrical and fluid flow controls for the foregoing apparatus. A cell 27 such as the cell of Fig. l is connected to have a reagent or electro-lyte conveyed therethrouyh in individual analysis quantities. A
pump 60 pumps air through a line 61 from a reagent container 62. A reagent valve 63 controls flow of the reagent to cell 27.
Referring to Fig. 2, the reagent flows into lower channel 49 and thus into and through the cell 27. Another fluid line ~
65 is positioned to carry the reag2n-t or other liquid from the cell 27 past an optical sensor 68 such as, for example, the sensor shown in Fig. 5. Line 65 then conveys the liquid to a drain container 69. A vacuum line 70 returns to pump 60. Thus ' the flow of the liquid through cell 27 is into the bottom of the cell and out through outlet 39 positioned above the cell. Preferably inlet channel 38 in cell 27 ~ill be loca~ed slightly ab~ve outlet channel 39 so that liquid normally will flow out channel 3~ rather than channel 38~
For analyzing a sample the sample is dissolved in a solvent to Eorm an el.ec-trolyte or reagen-t. By way of example, for tes-ting for i.ron in blood or serum a small sample of blood or serum, typically a 5 to l00 microliter sample is added to an electrolyte or chemical reagent which releases iron from its serum bonding and separates the transEer potentials of iron and its most usual interferring element, copper. .
If total iron-bindiny capacity is being measured, the serum is first fully saturated with iron, as by mixing it with an .
iron-containing ion exchange resin.
Preferably, the electrolyte or reagent for treating serum to release iron for testing comprises strong hydrochloric acid, e.g.
between about 5 l/2 and about 8 l/2 Formal, and preferably about 7 Formal, in a lower alcohol such as propanol or isopropanol~
Methanol and ethanol have been found nearly as effective as pro-panol or isopropanol, kut have the disadvantaye that they are more expensive, and they are more volatile and therefore more difficult to.handle. Higher alcohols such as butanol and the like are oper-able, but are less compatible with strong hydrochloric acid.
Other materials such as acetonitrile and acetone are also operable but are less satisfactory partly because o less satisfac~
tory performance and partly because of cost, volatility, .
toxicity and the like~ The apparatus is calibrated in accordance w1th the selected strengths of the hydrochloric acidO

1~L3QO(~3 T~e use of strong hydrochloric acld in lower alcohol as an electrolyte or reagent to release iron from its serum bond ing and -to separate the transfer potential o~ iron and its most usual interferrlng substance~ i.e. copper, in order to prepare the blood or serum for electrochemlcal analysis for iron is believed novel. .
In place of hydrochloric acid there may be employed other compounds having a high chlorine or halogen content, but ~uch other compounds have not been ~ound to be fully sa-tisfactoryO
For example, lithium chloride is a more expensive source of chloride ion and also tends to precipitate at least a portion of the serum. Hydrogen bromide is another source of halogen ion but is also more expensive and is notably more difficult to .
work with and is corrosive.
Included in the reagent or electrolyte is an extremely minute quantity of silver ion in the r-ange of about 200 parts per mil~
lion which assists in the operation of the silver reference electrode segmen-t 45. The reference poten-tial is the silver ion .
potential, maintained by reference electrode segment 45.
Accordingly, the reagent or electrolyte preferably will include 7 Formal HCl in propanol together with 200 parts per million silver ion and such reagent or electrolyte will release iron from serum or its iron binding components to make the iron avail-able to electrochemical measurement and also will permit separa- .
tion of the charge transfer potentials of iron and copper~ and give reproducible results in the analysis of serum iron by .
electrochemical measurement techniques in microliter sample quantities.

~ ~l 3~ 3 The prepared sample comprising a se~um to be tested, toyether with a measured quantity of a reagent or electrolyte is charged to cell assembly 27, and stirring commenced.
A potential control 70 applies two different electrical poten-tials 72 and 73 to two of the active testing electrode seg-ments, e.g. electrode segments 42 and 43. Electrical potentlal 72 ..
is set at a value which causes an electrochemical reaction of both i~on and copper, while electxical potential 73 is set at a value which causes electrochemical reaction of copper alone, as will be described in detail hereinafteru A reference potential is applied to the silver electrode segment 45, and another poten-tial is applied to counter electrode segment 44 and provides a source o current to the cell. Alternatively counter electrode .
segment 44 may be held at ground. The current or siynals from :
first and second testing electrode segments 42 and 43 are fed v~
to a logic module which subtracts the first signal from the second and, if desired, applies a multiple for calibration pur~
poses. By way of example, the current or signals from the two active electrodes segments in the cell 27 can be fed to a current .
convertor subtractor 75 with two variable gains or adjustment. The signal then goes to a signal accumulator 76, and then to a calibration blanking circuit 77 which also has a ' variable gain or calibrator 78. The signal from the calibration blanking circuit 77 then is ~ed to a readout 80 and~ in turn, to an autoblank control 81. The signal from the autoblank control is returned to the calibration blankin~ ircuit 77.

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~en the calibration is correct, an autoblank set 82 is operable to f.ix the circuits.
The electrochemical reactions which take place and are measured by the apparatus are the reduction of ferric ion to ferrous ion, the oxidation of ferrous ion to ferric ion, and the reduction of cupric ion to cuprous ion. (Generally, material will no~ be deposited on the active electrode segments, . .
and accordingly these reactions may be considered to be "charge transfer" .rather than electrolytic or electrodeposition reactions).
At active electrode segment 42 there occurs the reduction of ferric ion (Fe~3) to ferrous ion (Fe+2) and the reduction of cupric ion (Cu ~) to cuprous ion (Cu~l)O At active electrode segment 43 there occurs the reduction of cupric ion to cuprous ion and the oxidation of ferrous ion to ferric lon. As a rnatter of choice, .
active electrode segment 42 i5 set at the higher potential.
The signal~at the one electro~e is subtracted from the o~her with the following result:
( ) +3 ~ ~ Fe ; Cu~2 + - Cu and ~2) .
(B) Fe ~ Fe + e ; Cu ~ Cu - e. (3) by ~ubtraction .
(A) - (B) = Fe 3 and Fe ; Cu ~ n . (4) As can be seen, ~he reduction of cupric to cuprous ion i5 cancelled out in the logic with the result that the total of iron content is the signal which is fed.to the digital or other -readout.

~3~ 3 Gellerally, the po~en-tial on active electrode segment 42 may be varied betweel- about 0 to 1 volt while the poten-tial on active electrode segmen-t 43 may be varied between about 0 to 300 millivolts from that of segment 42. For testing for serum iron in accordance with the foregoing technique active electrode segment 42 will be set at a potential of about 460 millivolts while active electrode segment 43 will be set at a potential of about 250 millivolts It is to be appreciated that the invention is not limited to ~he measurement of serum iron, but that any electro-active substance may be detected and measured using -the foregoing process and apparatus. By way of example, the electrochemical measuring system of the present invention may be used for detecting and measuriny heavy metals such as zinc, cadmium, lead, copper, bismuth, gold, silver and thallium in blood samples.
As is well known in the art such heavy metals normally are com-plexed with blood, and thus must be released before they can be measured. A number of reagents are known in the art and are ;
available commercially for r~leasing such heavy metals from human blood~ One such reagent is called METEXCHANGE~
and is available from Environmental Sciences Associates, Inc., of Bedford, Massachusetts~ The manufacturer describes this reagent as comprising a dilute aqueous solution of calcium chloride, chromium tri-chloride, hydrogen ion, phosphate ion, aietate ion and a dispersing agent. The mixture of calcium ion and chromium ion is said to cause release of complexed heavy ESA-117 CIP ~22-metal in hlood so t~lat the total concen-tration of heavy metal can be effectively measured.
Moreover, the invention is not limited to the detection and measurement of heavy metals in biological samples. For example, heavy metals complexed with gasoline can be detected and measured in accordance wi-th the foregoing by dissolving gasoline samples in a reagent which comprises a dilute mi~ture of ICl, NaCl, N2H4HCl and a polyalcohol. The same reagent can be used to release various other heavy metals from a wide variety of organic samples. Other reagents which contain a metal ion which will displace the heavy metal of interest from the complex can also be used.
Additionally, a large number of organic substances are elec-troactive and thus can also be detected and measured in accordance with the foregoing invention including:~ unsaturated hydrocarbons, azides, triazines and phenothiazines, amino acids, amines and amides, phenols, aroma-tic OH, quinolines, quinones, imines, ' olefins, ketones, aldehydes, esters, and olefinic esters, ethers, organometallics, diazo compounds, nitro compounds, and halogens. The same reagents which are useful for dissolving these organic substances for liquid chromatography generally can also be used as the reagent in the process of the present invention.
Amongst suitable reagents are mentioned: water, lower alcohols, such as methanol, ethanol and isopropanol, and mixtures thereof.
If required a strong inorgani~ acid such as hydrochloric acid . . .

~3~ 3 or phosphoric acid, a strong base 5uch as sodium hydroxide, or a salt such as sodium chloride may be lncluded in the reagent to release the species oE interest from a complex.
For example, Eor analyzing blood samples for the presence of Tylenol, morphine or heroin in accordance with the present inven-tion a suitable reagent comprises methanol/water/phosphoric acid mixture comprising about 30% methanol, 0.1 to 1% phosphoric acid, and the balance water. For analy~ing blood samples for essential trace elements such as zinc, an aqueous solut'on of calcium acetate buffered to pH 3 has been found to be a suitable reagent. A normal saline reagent may be used to measure glucose in blood or serum.
The electrochemical measuring system of the present invention may also be advantageously employed for detecting and measuring substances such as cyanide, halogen, S02 and N0 . . ~.R.~ '.~. ~ . . X
in biological samples, water or sewage. The electrochemical measuring system of the present invention may also be adapted for use in monitoring of electroactive substances in chemical process streams. The required electrode potentials are approxi-mately the same as would be employed in controlled potential coulometric stripping of the same organic substances.
The extreme sensitivity of the electrochemical measuring system of the present invention permits accurate measurements in picogram region. Thus, the electrochemical measuring system of the present invention may be advantageously employed I

for making soil a~alysis for agricul-tural purposes and may also be used for metal prospecting. In regard to this latter feature, the process involves measuring soil and/or water samples taken in a yrid pattern in order to zero in on significant deposits of selected metals. By way of example, to zero in on deposits of relatively rare metals such as molybdenum, tungsten, vanadium, titanium and uranium soil or water samples taken on a grid are extracted with and analyzed in a reagent comprising alcoholic HCl solution such as a 20% solution of methanol in HCl.
The electrolyte is then charged to the cell, one of the active electrode segments is set at an electrical potential to oxidize the metal of interest while another of the active electrode segments is set at an electrolytic potential to oxidiza the metal of interest plus other interferring metals. The required electrode potentials are approximately the same as would be employed in con-trolled potential coulometric analysis of the same metal or metals. Other me-tals may be measured by changing the electrode potentials and/or the reagen-t. For example, for chromium a preferred reagent is alcoholic hydroxide solution such as 0.8 normal NaOH in methanol. The use of an alcoholic HCl solution as reagent for electrochemical analysis of molybdenum, tungsten, vanadium, titani~ and uranium, and the use of an alcoholic hydroxide solution as reagent for electrochemical analysis of chromium are believed novel.
Gaseous samples and/or airborne samples can also be analyzed by bubbling the gas or air through a suitable reagent to dissolve the substance of interest. The electrolyte can then be charged to the electrochemical cell as above described, and measurements made in accordance with the ~oregoing.
One skilled in the art will rec~nize that the invention is susceptible to modification. Thus, sensing electrode 34 in ~ 117 C~P -26-~ ~3~

acco~dance with the present invention has been shown as comprising t~o active testlng elec~rode segments, a reference electrode seg-ment and a coun-ter electrode segment with the electrical potentials on the two active testing electrode segments being adjusted accord-ing to the particular substances being detected and measured.
One skilled in the art will recognize, however, that electrode 34 may comprise a large numbPr of active testing electrode segmen-ts, e.g. electrode 34 may comprise 50 or l00 electrically discrete active testing electrode segmen-ts, each segment being electrically connected to a different electrical potentiaL to effectively re-produce an entire current voltage curve. For example, the elec-trode 34 may comprise twelve active testing electrode segments at a series of electrical potentials, which may be 20 to 80 millivolts offset. Thus, to electrochemically analyze samples which may contain a variety of electroactive substances of inter-est in whic~ there are known or suspected interferring su~stances, L. 3~ 4 3 it is a simple matter to store the signal information from each electrode seg~ent and to select out or sort only those active electrode se~ments whlch are at the particular electrical poten-tials which produce the clesired electrochemical reactions, derive signals from those electrochemical reactions, and sum (add or subtract) the signals to arrive at the desired measurement. The selected active electrode segments may be connected in manually ..
by the operator, P.g~ according to printed instructions. .Obvious-ly, such an apparatus may also include a plurality of reagents, supplies, reagent valves, etc. so that a particular reagent may be introduced depending~on the particular substance being detected and measured.
The foregoing apparatus has been described as belng run under operator control; however, the apparatus can be made to operate automatically as follows:- Reerring to Fig. 6, a cGntr ,, .

.

JI~3~ 3 synchronizer 85 is providecl for ac-tuating a pump and valve timing control 8G and also an ~nalog tilning control 87. The analog tirnin~ control 87 ls in the ready position and is activated for analysis by a start analysis control 88 which appears on the apparatus as start test button 24.
Optical sensor 68 whose operation is illustrated in Fig. 5 directs a signal to flow sense circuit lg which in turn sends a signal to pump and valve timing control 86 and analog timing control 87. Should the flow through line 65 be inadequate fox complete flushing of cell 27, the signal from Elow sense circuit 90 operates to turn off pump 60 or close valve 63 or both, and to inactivate analog~timing control 87 so that an analysis cannot be started without resetting the apparatus.
A power supply 91 operated from an A/C power source 92 sup plies a positive voltage through line 93, a negative voltage through line 94, and a ground potential through line 95 which are supplied to the cell potential control 70. The cell poten- ¦
tial control 70 car. be controlled by potential set 96.
In a preferred form of automatic controls the apparatus con-sists of two sections: analog circuitr~ for converting, condi-tioning and displaying electrochemical signals; and reagent hand-ling circuitry for automatic sample handling.
The analysis cycle is controlled by two sequential timers 8l7.
The first timing interval (30 seconds) is initiated after the start analysis switch 88 is depressed. This sequence is used to bring the cell to equilibrium. The second interval (20 seconds) is the concentration measurement. During this ~lme the electrochem~
ical signal is converted and displayed. In a preferred farm the , ' ' ~13~3043 apparatus displays the "count down" or "count up" digitally during the measuremen-t. Cell re~erence potential is controlled by potentiastat circuit 70 and is set by control 96.
This potential is applied between the reference electrode segment 45 and active electrode segment 42. A difference potential is seen between active electrode segment 43 and reference electrode segment 45. This difference potential is set by offset 2 control operating on current convertor subtractor 75. The equivalent potential becomes [Eset 1 Eoffset~
During ~he measurement interval the cell currents are fed into current-to-voltage convertor circuit 75 and gained con-trolled by potentiometers "Gain 1" and "Gain 2". The difference of the resulting voltages is taken and fed into the accumulator circuit 76 and integrated during the measurement interval. The integrated voltage then has the "autoblank" value subtracted ~.r_. ~ ,.
from it and gained by calibrate circuitry 77.
The resultant value is then displayed on the readout 80 in direct units of micrograms of iron per lOOml (~g%) of serum. -~hen the digital display stops counting the reagent or electrolyte containing the sample is flushed into container 28 and a new supply of reagent or electrolyte is introduced into the cell from container 29. When the start test indicator 24 lights up again, , the apparatus is ready for a next sample. The entire test may take less than one minute, the largest portion of which is the pre- -~
liminary mixing time.

l:t.3~091~3 Reagent or electroly~e can be automatically charged to the cell in a number o~ ways. One way is to automatically fill the cell when the uni-t switches from the standby to run position; ano-ther way is to automatically fill the cell at the end of each ana-lysis cycle.
Pump and valve timers are set "on" by the control synchronizer 85 from a trigger signal received by the standby control switch or the analysis cycle timer. The solenoid valve 63 is used to control reagent flow into the cell. A pump supplies nominal pressure (e.g. 4 psi) to reagent supply 62 and a nominal e.g.
vacuum (l7" Hg) to drain reservoir 69. The pressure forces clean reagent through the valve into cell 27. This increase in cell volume i5 taken off through the drain line to the drain reservoir 69. The reagent inlet valve is timed on for a short time, e.g.
~ seconds, and the pump is left on for an additional 2 seconds to .,,.. ~. ~ , , ..
drain any excess reagent above a set level from the cell.
A flow sensor 68 consisting of optical sensor 56 and ~low sense circuit 90 monitors the cell drain line 65 during the reagent flushing cycle. If there is no reagent flow or if a low amount of reagent passes through the cell, the flow sense circuit 90 will reset the pump and valve timers and thus prevent the start of an analysis. An audio alarm and indicator light I
(light 23) may also be ac~ivated at this time. Thus, a new cycle cannot be started until the operator places the instrument in the^
standby condition which resets the flow sense circuit 90.

The flow sense circuit 90 comprises a~ optical sensor (LED 55 and phototransistor assembly 56, Fig. 5) and is placed a-t the cell drain line. In operation, the output Erom flow sense circui~ 90 changes from a low voltage level (line emp-ty) to a higher voltage level (reagent flowing). This level change is sen-sed and integrated during the first 4 seconds of the reagent cycle.
If the integrator voltage is below a preset level at the end of the 4 second interval, instrument lockout is activated.
In the autoblanking operation, when a blank conecntration reading is taken and is to be nulled out of future readings, the unit switched from "run" to"autoblank". The autoblank set switch is depressed, starting a 4 second timer. The binary coded decimal output from the display latched in the circuit.
This BCD number is then converted from a digital to an analog signal.
An analog voltage of correct polarity and magnitude is ESA-117 CIP ~32-3()43 fed to the calibratlon circuitry and subtracted from the con- .
centration analog voltage resulting in a zero output to the display.
Alternatively, the apparatus may be made to operate auto-matically, e.g. by means of switching using a microprocessor.
In such case, for a known substance, a tape containing instrument .
instructions would be inserted in the microprocessor, which then selects the reagent to be added to the cell, and the electrode potentials. The results could then be displayed for visual obser- - .
vation as on a CRT tube or printed out, or the result, may be read.
into memory for appropriate mathematical manipulation and then displayed. For an unknown substance, the instrument could be constructed to connect a plurality of electrically di~crete .
active testing electrodes at different electrical potentials to thus reproduce an entire curre~t voltage curve which can then be compared to current voltage curves for known electroacti,e -pecies.

:
.
.

: : ' .

The identification of the unknown species can be determined by ma-tching curve sh~pes while the amount of an electroactive species presen-t in the sample can be determined ~rom the area under various sections of the curve for the unknown. More specifically, Fig. 7 illustrates a typical current versus potential chart obtained in accordance with this invention. In this graph, the horizontal axis indicates the difference potential, in volts, of working electrodes at increasingly more positive potentials with respect to the silver/silver chloride reference electrode. The vertical axis represents the anodic current, in microamperes, at the indicated potential. The waves of the current versus potential curves indicate a sharp change in current due to the change in concentration of each electroactive species as it reacts in the reagent. Since the potential at whi.ch a particular electroactive species reacts is characteristic of a particular .

species in a particular reac3ent, the electroactive species present in the sample are readily identi.fied. Also, since the presence of any interferring electroactive species is cancelled out by the electronics, the areas under the peaks are directly related to the total amount of and thus to the concentration of each electroactive species in the sample solution.
A feature and advantage of the present invention is ~.hat .
electrochemical measurements are made of charge transfer reactions substantially simultaneously with the occurrance of the reactions.
Thus electrochemical measurements in accordance with the present invention can be carried out simultaneously on more than one sub-stance of interest in a sample by application of suitab1e electrical~
potentials on the various active electrode segments, and through signal sorting. For example, a blood sample may be tested simul-taneously for lead and chromium.

Various other changes will b~ obvious to one skilled in -the art. For example, the actlve electrode segments have been illustrated as comprlsing continuous rings or bands; however, one skilled in the art will recogni~e that the active electxode segments may comprise individual dots or segments, or a series of dots or segments. Moreover, while the electrode preferably comprises a hollow cylindrical, the similar advantages may be achieved by shaping the electrode as a hollow cone and by pro-vi~ing a stirrer of mating size and shape. Furthermore, one or more active electrode segments may be added to change the electrode area at selected voltage potentials so as to reduce or null out otherwise very large signals from interferring electro-active species, or reagent signals in the case of very dilute solutions, and thus increase sensitivity to a particular electro-chemical species of interest, and to balance signals. Moreovex, ,.~,............. ~ , ., neither the reference electrode nor the counter or power elec trode need be mounted as segments or electrode 34, but can be separately provided in known manner in contact with the solution being measured. For example, the reference electrode and/or the counter electrode may be formed in plug 46. Also, the apparatus -may comprise more than one reference electrode and/or more than one counter electrode. The apparatus could also be adapted to operate as a flow cell to thus provide a continuous profile of a process.

In addi~i~n to the Eoreyoing, it will be understood, as shown in Fig. 8, that the electrochemical system may comprise a pair of side-by~side cell assemblies 27A and 27B. Cell assemblies 27A and 27B are similar to cell assembly 27 as above describ~d. In this latter case one of the cell assemblies, e.y.
assembly 27A is designed to be the analysis cell while the other cell assembly 27B is a blank correction cell. Each active electrode segment in cell assembly 27A is paired with a corresponding active electrode segment in cell assembly 27B at the same potential. In use sample containing reagent is injected into cell assembly 27A while pure reagent is injected into cell assembly 27B. The cell contents are stirred at substantially identical rates, and charge transfer signals derived as before. The signals from the active cell segmen-ts in the two cell assemblies 27A and 27~ are summed,~ e.g.
as by subtracting the signals derived from cell assemhly 27B from the signals derived from cell assembly 27A, with the result that all background signals are essentially nulled. An advantage of employing two similar cell assemblies is that signals resulting from impurities in the reagents are nulled. Also, settling effects resulting from a change of reagent, cleaning cycles, etc., are also nulled. Cell assemblies 27A and 27B may comprise identical active electrode are-s, or one of the oell asse ~ lies, ~typically ~3~

th~ blank correcti~n cell assembly 27B) may be made smaller -than the analysls cell assembly 27A and the differences in active electrode areas compensa~ed electrically in known manner. As men-tioned above, the stirring rate in cell assembly 27A should be sub-stantially identical to the stirring rate in cell assembly 27B.
The simplest way to assure matching is to mechanically connect the stirring means 50A and 50B in the two cell assemblies 27A and 27B to a single motor.
Alternatively, the two ~ell assemblies may be stacked one on top of another, e.g. as shown in Fig. 9 at 27C and 27D, and the cell contents stirred by a stirring means 50C and 50D
which are mounted on a common shaft 51A. Obviously, care must be taken to prevent fluid transport between the two cell assemblies 27C and 27D. This can be assured by close manufacturing tolerances and with sealing means as are well known to one skilled in the art. As before, grooves 52C and 52D are provided on stirring means 50C and 50D, respectively. If desired, these grooves may be made to run in opposite directions to one another to minimize fluid transport batween cell 27C and 27D.
A particular feature and advantage of the present invention which results from the use of an electrode having a plurality of active testing electrodes at diffexent potentials in accordanc'e with the present invention is the elimination of capacitance signals which were inherent in-prior art electrochemical measuring in which the potential on an electrode is changed to obtain a m~asurement.
Still other features, advantages and objects will be obvious to one skilled in the art.

ESA-117 CIP -3~-

Claims (13)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. An electrode for electrochemical testing adapted to measure simultaneously at least two electrolytic potentials with reference to a reference potential, comprising:
a hollow cylindrical electrode body of an insulating material, open at least at one end;
a smooth cylindrical inner surface on said electrode body;
at least a first, second, and third electrode segments mounted at the inner surface of said electrode body and having active surfaces flush with the inner surface of said body, each of said segments being electrically insulated from each other electrode segment and separated therefrom by said insulating material;
one of said electrode segments being a reference electrode; and means to connect each of said electrode segments to a different electric potential.

2. An electrode according to claim 1, having first, second, third and fourth electrode segments;
said first and second segments being test electrode segments adapted to measure simultaneously two electrolytic potentials;
said third segment being a reference electrode segment;
said fourth segment being adapted to provide an electrolytic current source different from the potentials on said first, second and third segments.

3. An electrode according to claim 1 or 2, wherein said reference electrode comprises silver, and said first, second and fourth electrode segments comprise carbon.

4. An electrode according to claim 1 or 2, wherein said reference electrode has at least its surface formed of a reference metal.

5. An electrolytic cell for electrochemically testing a sample in solution, said cell comprising:
a sensing electrode as claimed in claim 1, in the form of a hollow cylindrical body defining an enclosure for holding said solution for testing, said body being (a) formed of an electrically insulating material and (b) having a substantially smooth cylindrical inner surface;
at least two active testing electrode segments mounted at the inner surface of said body, said electrode segments (a) having electrically active surfaces which are substantially flush with said smooth inner surface and (b) electrically insulated from one another by said insulating material;
a counter electrode having an electrically active surface mounted in said enclosure;
a reference electrode having an electrically active surface mounted in said enclosure; and, means for connecting (a) said at least two active testing segments to different electrical potentials, (b) said reference electrode to a reference potential, and (c) said counter electrode to yet another potential.

6. An electrolytic cell according to claim 5, wherein said reference electrode comprises one or more electrode segments mounted at the inner surface of said body, each of said reference electrode segments (a) having an electrically active surface substantially flush with the inner surface of said body, and (b) being electrically insulated from one another and from all other electrode segments on said body.

7. An electrolytic cell according to claim 5, wherein said counter electrode comprises one or more electrode segments mounted at the inner surface of said body, each of said counter electrode segments (a) having an electrically active surface substantially flush with the inner surface of said body, and (b) being electrically insulated from one another and from all other electrode segments on said body.

8. An electrolytic cell according to any one of claims 5 to 7, wherein said reference electrode comprises silver, and said active testing electrodes and said counter electrode comprise carbon.

9. An electrolytic cell assembly comprising first and second electrolytic cells as defined by any one of claims 5 to 7, said first electrolytic cell being adapted to hold a sample in a reagent and to measure charge transfer reactions of said sample and said reagent, said second electrolytic cell being adapted to hold said reagent alone and to measure charge transfer reactions of said reagent alone, said first and said second electrolytic cells having corresponding active electrode segments, and means for pairing electrode segments in said first electrolytic cell with corresponding electrode segments in said second electrolytic cell at the same potential.

10. Apparatus for electrochemical testing a sample in solution to identify selected substances therein, said apparatus comprising at least one electrolytic cell as defined by claim 5, and means for comparing signals corresponding to electric current flow at at least two active testing segments to determine, by said comparison, the quantity of said selected substances in said sample.

11. Apparatus according to claim 10, including a) stirring means mounted in said sensing electrode;
b) sample input means for introducing a measured quantity of sample into said sensing electrode;
c) input means for introducing electrolyte liquid to said sensing electrode;
d) outlet means for discharging spent sample and/or spent electrolyte from said sensing electrode; and e) means for supplying electrolyte liquid to said input means.

12. Apparatus according to claim 10 or 11 for electrochemical testing of serum iron, wherein one of said electrode segments is adapted to measure a first charge transfer for both iron and copper, and another of said electrode segments is adapted to measure a second charge transfer for a different quantity selected from iron and copper, and including means for applying to said one electrode segment a first potential for measuring the charge transfer of both iron and copper; means for applying to said another electrode segment a second potential for measuring the charge transfer of a different quantity selected from iron and copper;
and means for comparing signals corresponding to said first and second charge transfer measurements to determine by said comparison the quantity of iron in said sample.

13. Apparatus according to claim 11, and including means for flushing electrolyte through said cell to remove electrolyte therefrom and to replace it with fresh electrolyte, said means for flushing comprising a first conduit for supplying fresh electrolyte to said cell, a second conduit for receiving spent electrolyte from said cell, an optical emitter and an optical detector on opposite sides of one of said first and second conduits, and curved transparent walls in said one of said conduits between said emitter and said detector, thereby radiation from said emitter is more fully focused on said detector while electrolyte liquid is in said conduit.