A HYDROGEN ELECTRODE VESSEL.’

BY Wiw. MANSFIELD CLARK.

(From Ihe Research Laboratories of the Dairy Division, Bureau of Animal Industry, United States Department of Agriculture, Washington.)

(Received for publication, October 15, 1915.)

INTRODUCTION.

With the extension of the use of the hydrogen electrode, espe- cially in biochemistry, have come numerous modifications of the classic type of electrode vessel. The form here described was developed to meet some special requirements in a study of the hydrogen ion concentrations of bacterial cultures, and, although it embodies few radical improvements, its convenience and suc- cess are offered as sufficient apology for adding its description to the many now found in the literature.

The classic method of operating, in which hydrogen is bubbled through the solution in which a platinized electrode is wholly or partially immersed, proved useless in the studies referred to. With some culture media, even when electrode and solution were previously and separately saturated with hydrogen as recom- mended by Desha and Acree,2 constant potentials were not ob- served until the lapse of several hours, during which period great changes may occur in active cultures. A rapid as well as accu- rate method was therefore demanded.

A quick attainment of constant potential, even in blood, has been shown by Michaelis and Rona to be obtained if the plati- nized electrode, previously saturated with hydrogen, is allowed merely to touch the surface of the solution. This is probably due, as suggested by IIasselbalch,4 and again by .Konikoff,5 to a

1 Published by permission of the Secretary of Agriculture. 2 Desha, L. J., and Acree, S. F., Am. Chem. Jour., 1911, xlvi, 638. 3 Michaelis, L., and Ronn, P., Biochem. Ztschr., 1909, xviii, 317. 4 Hasselbalch, 1~. A., B&hem. Ztschr., 1913, xlix, 451. 5 Konikoff, A. P., Biochem. Ztschr., 1913, Ii, 200.

475

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476 Hydrogen Electrode Vessel

rather sharply localized equilibrium at the point of contact. Re- ductions and gas interchanges having taken place within the small volume at the point of contact, diffusion from the remaining body of the solution is hindered by the density of the surface layer with which the electrode only comes in contact.

In exploring new fluids it appeared to the writer precarious to rely upon such a device, which appears to take advantage of only a localized and hence a pseudo-equilibrium, and which makes no allowance for a possible difference between the solution and surface film in the activity of the hydrogen ions. Hasselbalch’s principle seemed therefore to be more suitable.

Hasselbalch6 found that a very rapid attainment of a constant potential can be obtained by shaking the electrode vessel. Un- der these conditions there should be not only a more rapid inter- change of gas between the solution, the gaseous hydrogen, and the electrode, an interchange whose rapidity Dolezalek? and Bose8 consider necessary, but the combined or molecular oxygen, or its equivalent, in the whole solution should be more rapidly brought, into contact with the electrode and there reduced. Fur- thermore, by periodically exposing the electrode the hydrogen is required to penetrate only a thin film of liquid before it is absorbed by the platinum black. The electrode should therefore act more rapidly as a hydrogen carrier. For these reasons a true equi- librium embracing the whole solution should be rapidly obtained with the shaking electrode; and indeed a constant potential is soon reached.

In Hassclbalch’s design t,hcre appeared to the writer to be cer- tain objectionable features. Aside from the rather clumsy dome, which Pauli” and also Manabe and Matula have replaced with a stopper, Hasselbalch’s design and the various modifications which have since appeared maintain the long axis vertical. This, if complete rotation is not resorted to, requires a very wide angle of rotation to gain a maximum exposure of liquid surface. More

6 Hssselbalch, Biochem. Ztschr., 1911, xxx, 317; 1913, xlix, 451; Compt. wnd. tmv. de Lab. Cadsberg, 1911, x, 69.

7 Dolezalek, F., Ztschr. f. Electrochem., 1898-99, v, 533. 8 Bose, E., Ztschr. f. ph,ysikal. Chcm., 1900, xxxiv, 701. !J Pauli, W., cited by Manah, I<., nnd Alntuln, J., Biochem. Ztschr.,

1!)13, lii, 369.

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W. M. Clark 477

objectionable is the persistence in most of the published electrode vessels of dead spaces, especially in the tubes adjacent to the body of the vessels. Acree and Myers lo state that constant potentials cannot be obtained so long as the portion of the solution used to connect with the KC1 is left unsaturated with hydrogen.

It is unnecessary to speculate upon the quantitative signifi- cance of these and other errors in design, since they arc easily eliminated as the following design will show.

The Vessel and Its Operation.

Ey setting up the chain in an air bath there are avoided many of the complicating details of design necessary in an oil bath. Theoretically an air bath is to be preferred if it can be kept at a fair degree of constant temperature, since air, because of its low specific heat, exchanges heat slowly wit.h the objects it bathes. The purpose of a constant tcmperaturc bath is to keep not itself, but the apparatus it contains, at constant temperature. An air bath may bc permitted to fluctuate in temperature more widely t,han an oil or water bath with the certainty that, if the fluctua- tions are regular and periodic as well as moderate, 6he appara- tus will lag and remain very constant. The air bath used is well insulated with two inches of cork besides its well constructed frame and compo-board lining. Active as well as smooth circula- tion of the air is accomplished by fanning with a Firocco fan from one end of a cent,ral box containing the apparatus, and by using the space hctwccn this box and the bat.h walls as flues for the re- turn of the air to the other end. An ordinary fan is worthless in an air bat,h if smooth circulation is demanded. The heating unit is a length of hare nichrome No. 30 wire which retains very little heat when the current is stopped. The regulator is a grid containing about 60 cc. of pure mercury with the regulator head described in a previous paper.” When the regulator was first used periods were observed in which the temperature remained const,ant for periods of 8 hours within * 0.002” (tapped Eeck- mann thermometer reading). This was a suspicious constancy. It soon settled down to about * 0.05, but’ the variations from moment to moment were found by an eight pair copper-constan-

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478 Hydrogen Electrode Vessel

tine therm0 element to consist of very regular fluctuations of only * 0.003, about a very slowly drifting mean. The present regu- lator has been in oontinuous use for a year and a half without attention.

In the manipulation of the vessel the purpose is first to bring every portion of the solut.ion into intimate contact with elec- trode and hydrogen atmosphere, and then to draw the solution into a wide tube in forming the liquid contact.

L! FIG. 1. Sectional view of the vessel an4 its accessory parts.

The electrode vessel is first flooded with hydrogen, of which there should be an abundant flow. Then, with the vessel rocked back until cock A is at its lowest point, the solution to be tested is run in from the small container B. It displaces hydrogen which wastes through C at D. Cock C is closed when the vessel is about + full of solution, and -4 is then turned as shown in the figure to allow a constant atmospheric pressure of hydrogen to bear upon the solution. The vessel is now rocked so that the electrode is alternately exposed to hydrogen and immersed in the solution.

I1 Clark, W. M., Jour. Am. Chem. Sot., 1913, xxxv, 1889.

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W. M. Clark 479

The rocking of the vessel is accomplished by supporting it upon a bar which is pivoted behind the flexible tube E and which rests in a groove of the eccentric wheel F. By changing the position of this wheel relative to the pivot of the bar the vessel can be given excursions of various ranges. The range should be wide enough to immerse and expose the electrode completely, and the speed of rocking should be such that the liquid actively but smoothly surges from end to end.

After the vessel has been rocked a sufficient length of time the solution must be brought into contact with the potassium chloride solution which completes the chain between the electrode and the calomel cell G. Before making this contact fresh KC1 solution is allowed to flow from the reservoir H and waste at D. The flow is stopped by closing cock I. The flexible rubber tube E is then pinched; and, after turning C to connect with the vessel, the pinch is released. This brings the liquid contact below the constriction of the cock and makes it occur in a wide tube in ac- cord with the recommendation of Cumming and Gilchrist.12

In the diagram the vessel and cocks are shown in the positions they occupy during a potential measurement. It will be noticed that the only cock in the chain which is closed is I. It is n-ces- sary to keep one cock closed to prevent the various solutions from being displaced from their proper positions. Cock I is chosen because it occurs in the best conducting liquid and does not sepa- rate dissimilar solutions. It should be left ungreased in the cen- ter; but, to prevent the creeping of KCl, and to insure ease in turning, the key is touched with vaselin at its widest part and the socket at its narrowest part. Then when the key is replaced it will ride in two rings of grease and with the central portion un- contaminated and filled with a good conducting film of potas- sium chloride solution.

In the specifications of this vessel it was demanded that the glass blower seal the cocks A and C to the body of the vessel as closely as possible and without constrictions at the junctures. When the stopper holding the electrode is forced down further than shown in Figure 1, the interior is left with practically no dead spaces for liquid or gas. By the use of the cocks the liquid within

1% Gumming, A. C., and Gilchrist, E., Tr. Faraday Sot., 1913, ix, 174.

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Hydrogen Electrode Vessel

the vessel is sharply separated from that without and no inter- change can occur.

With the long axis horizontal the vessel needs only to be rocked slightly instead of rotated through a wide angle in order to expose a maximum solution surface and alternately expose and immerse the electrode. The gentleness of the required rocking not only prevents but tends to destroy the froth which is so easily produced in protein solutions.

With two such vessels mounted in parallel and connected through a three-way cock at I with the rest of the chain, determi- nations may be made with one while the other is being prepared for the next determination. This mounting also permits the measurement of one hydrogen electrode against another.

A word might be said here in regard to the operation of cock C. A study of the various positions of the key will show that if the portion of the bore which corresponds to the stem of a 1 is not filled before the liquid contact is formed it will remain un- filled and may give up a bubble of hydrogen which will be difficult, to displace. This may be avoided by the following procedure: In filling the vessel the key of cock C is kept in the positionl. Before liquid contact, is to be made I is opened and C is turned clockwise rapidly. With a little practice all the bores of the key are filled in this way without allowing any KC1 to flow into the vessel. Mr. G. E. Cullen has suggested a three-way cock at C with outlets set at 120”. This permits the use of a two-way key with obvious advantage.

With the exception of a few cases such as measurements of acetate-acetic acidmixtures there has been no occasion to object t.o t,he USC of a rubber stopper to hold the electrode. A glass stopper has not been used, partly because it would limit the choice of electrodes, partly to avoid the use of grease,13 and partly because of the difFiculty of grinding a stopper so that no capillary space will be left between its end and its socket to trap liquid. The rapidity with which measurements are taken reduces the influ- ence of solution of the stopper or indeed of the glass. If the rub- ber must be protected a light coat of low melting point paraffin, warmed when the stopper is forced into place, will do.

13 The surplus grease in each of the cocks was removed by a vigorous stream of solvent followed by alcohol and abundant water.

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W. M. Clark

An adequate discussion of the accuracies attained with this vessel would involve a lengthy argument upon the values and reproducibilities of the various sources of potential in the whole chain. In the work so far reportedl” there has been no occasion to enter into this argument, since, by every criterion applied by others, the potentials were judged to be accurate within a milli- volt, and no deduction based upon smaller differences has been presented. In this discussion we shall neglect corrections for barometric pressure, possible deviations of calomcl electrodes, and estimates of the values of contact potentials. Only the observed potentials will be given to show the precision attained at the hy- drogen electrode. The periods of measurement were too short and the hydrostatic pressure in the hydrogen generator too closely watched for variations in hydrogen pressure to affect the argu- ment,. Only one calomel cell was used in any one experiment and its deviation from other assumed standards was too small to consider. Pot,ential measurements were made with a Leeds and Northrup type I< potentiometer and the galvanometer cus- tomarily supplied with this potent,iomcter. The potentiometer was carefully calibrated by the Bureau of Standards. Two Bureau of Standards Weston cells furnished the known potential. The working standard was frequently compared with these, and the battery current was adjusted against this working standard at each potential measurement. The measured and measuring sys- tems were electrically shielded against stray currents.

It is to be noted in the first place that if liquid contact is made at once so that the potential of the chain may be measured while equilibrium is being attained within t,he vessel, the potential of the chain rises rapidly. This may be seen in Table I. That this rise is due to processes at the electrode is evident from the fact that the maximum potential is almost immediately observed if the vessel is shaken for a short period before liquid contact is

formed (Table II). In passing it may be said that the 1 per cent peptone solution used in these experiments has such a small buffer effect that the maximum observed variation in potential corre- sponds to that which would be produced by the addition of 0.0002 cc. normal acid or alkali to 10 cc.

I4 Clark, W. M., Jour. Med. Research, 1915, xxxi, 431; Jour. Infect. Dis.,

1915, xvii, 109. Clark, W. M., and Lubs, H. A., Jour. In.fect. Dis., 191.5, xvii, 160. Clark, Jour. Biol. Chem., 1915, xxii, 87.

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482 Hydrogen Electrode Vessel

In Table I a slight decline in potential is seen to have occurred after the maximum had been obtained. In this case the drift is thought to be located at the liquid contact and to be hastened by the disturbance due to shaking the vessel. In other cases the drift of the total potential of the chain has been toward a higher value. In every case this drift has been so slow, unless the liq- uid contact has been purposely and vigorously disturbed, its direction has varied so with different solutions, and its magnitude has been so small, that a clear tracing of its origin is difficult. It is believed to be located chiefly at the liquid contact. There- fore, in common with Lewis and Rupertls it is believed that better potentials are obtained directly after the formation of liquid con- tact.

Some experimenters continue observations over a long period and choose those potentials which remain constant during an ar- bitrarily selected central period. If under these circumstances there are drift.s at electrode and liquid contact of opposite sign, the selection of a central period of constancy may be merely the selection of a period in which the opposite drifts have become ap- proximately equal.

Since it is possible to reach equilibrium very rapidly at the electrode by the shaking method, and since drift at the liquid junction has been frequently reported by others and in some cases definitely traced while using the apparatus here described, it seems more reasonable to depend upon measurements continued for only a short time after the preliminary shaking, and to rely upon the reproducibilit,y of several such measurements rather than upon the record of a single long experiment.

In the accompanying tables are given a few examples of meas- urements made with this vessel. Particularly interesting are the measurements of a culture medium containing 10 per cent gelatin. This was a stiff gel at lower temperatures, but at 30.0’ it was sufficiently fluid to use in the electrode vessel. With no frothing of the solution satisfactcry potentials were obtained within a remarkably short’ t,ime. Equally satisfactory results were obtained with high fat milks.

So far there has been no occasion to investigate solutions of

15 Lewis, G. N., rind Rupcrt, F. F., Jour. Am. Chem. Sot., 1911, xxxiii, 299.

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W. M. Clark 483

low hydrogen ion concentration containing COZ. For such solu- tions the vessel should do quite as well as Hasselbalch’s, since its principles are the same.

In conclusion it must Abe admitted that no comparisons other than the one alluded to in the introduction have been made be- bween the vessel here described and other designs. Therefore no proof of superiority can be advanced for the new design. Occa- sionally in the course of several thousand potential measurements the results have not been as satisfactory as those reported in the tables. Occasionally results have shown better agreement. Those reported are fairly representative of the kind of results which careful manipulation will bring; and, when the time within which they were obtained is taken into consideration, it may be said that they are satisfactory for all ordinary purposes. Before closer agreement can have any significance a great deal of more fundamental work will have to be done upon several problems such as that of liquid contact potential.

TABLE I.

Showing rise in total potential during shaking. Chain: Hg HgCl [ Saturated KC1 1 1 per cent peptone solution 1 Pt Hz Liquid contact formed from the first. Shaking continued between

measurements.

Vesd

A

B .

Time. Potential.

min.

0 1 2 4 5” 9

11 13 17

0 1.5 4 6 8

10

volts

0.6490 0.6680 0.6729 0.6740 0.6740 0.6737 0.6736 0.6737 0.6735

0.6325 0.6653 0.6735 0.6737 0.6736 0.6734

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TABLE II.

Potential measurements of a 1 per cent peptone solution. Preliminary shaking 5 minutes. Vessel not shaken after the formation of liquid COII-

tact. Chain: Hg HgCl / Saturated KC1 / 1 per cent peptone solution j Pt If,

Vessel.

A. . :

B......

A. . .

B . . . . . . ..__........_...

Bverage.

Time.

min.

I 2 4 5

I 2 4 5

1 2 4 5

I 2 4 5

Potential.

vczts 0.6736 0.6738 0.6738 0.6738

0.6742 0.6742 0.6742 0.6742

0.6737 0.6739 0.6739 0.6739

0.6736 0.6738 0.6733 0.6738

0.6739

,TABLE III.

Potentials of the chain: Hg HgCl / Saturated KC1 / Borate-boric acid mixture j Pt H?

No shaking between measurements.

Vessel. Preliminary shaking. Time of measurement. Potential.

min. min. vczts A............... 5 1 0.80039

5 0.80040 12 0.80040

B.............. 7 1 (0.80025) 5 (0.80025)

A . . . . . 10 1 0.80030 20 0.80033

B 10 1 0.80034 15 0.80034

Average. 0.80036

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TABLE IV.

Potentials of the chain: Hg HgCl 1 Saturated KC1 1 Phosphate mixture 1 Pt H,

No shaking between measurements.

Vessel.

A.

A.

B . . . . . .

ilverage. . .

Prelimimry shaking.

min.

11

9

9

13

-

-.

_

_ .

-

Time of measurement.

min.

1 7

11

1 3

12

1 13

1 9

.-

-

Potential.

volts

0.64235 0.64240 0.64238

0.64239 0.64245 0.64240

0.64249 0.64245

0.64249 0 64250

0.64243

TABLE V

Potentials of the chain: Hg HgCl 1 $ KC1 1 Saturated KC1 I Gelatin culture medium 1 Pt HP

Preliminary shaking 10 minutes. No shaking between measurements.

Vessel. Time. Potential.

min. volts

A. . . . 5 0.7420 10 0.7417 15 0.7417

B . . . . 5 0 7411 10 0.7419 15 0.7419

TABLE VI. Potentials of the chain:

Hg HgCl 1 & KC1 1 Saturated KC1 1 Milk I Pt Hs Preliminary shaking 5 minutes. ,No shaking between measurements.

Vessel. I

Time. I Potentid.

min. volts -4. . . . . . . . . . . . 5 0.7304

10 0.7304

B . . . . . . . . . . . . . . . . . . . . . . 5 0.7303 10 0.7303

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486 Hydrogen Electrode Vessel

TABLE VII.

Potentials of the chain: Hg HgCl 1 & KC1 1 Saturated KC1 [ Meat infusion medium 1 Pt Hn Preliminary shaking 5 minutes. No shaking between measurements.

Vessel. Time.

min.

A . 1 5

Potential.

B . . . 1 3

13 28 35

volts 0.7014 3.7014

0.7013 0.7014 0.7014 0.7014 0.7013

Potentials of the chain: __

TABLE VIII.

Ilg HgCl 1 $ KC1 [ Saturated KC1 5 day culture of B. coli in Pt H, 1 per cent peptone 1 per cent dextrose I

Preliminary shaking 5 minutes. No shaking between measurements.

Organism. Time. Potential. -.

min. v;zts

hw..................... 5 0.5970 10 0.5970 15 0.5970

hr.. 5 0.5960 10 0.5960

hy 5 0.5937 10 0.5958 15 0.5958

hz 5 0.5947 10 0.5956 15 0.5956 20 0.5956

fg.. I.. . . . 5 0.5942 10 0.5960 15 0.5962 20 0.5962

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Wm. Mansfield ClarkA HYDROGEN ELECTRODE VESSEL

1915, 23:475-486.J. Biol. Chem. 

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