THE USE OF THE QUINHYDRONE ELECTRODE FOR HYDRION CONCENTRATION DETERMINATION

ON SERUM.*

13~ GLENS E. CcLLEl;t AKD EISAR I3IILKWS.

(From Ifedical Clinic A and the Chemi.stry Laboratory, Unirersitg o.f Copen- hagen, Copcnhagcn, Denmark.)

(Received for publication, April 28, 1925.)

The quinhydrone electrode, as developed by Biilmann (1, 2)) has proven a most convenient method for measuring the hydrogen ion concentration of solutions in many types of investigation. It is more convenient than the hydrogen electrode in ordinary solutions because special apparatus for the preparation and con- trol of hydrogen is not necessary. In COz-containing solutions it should bc especially convenient, for the greatest difficulties and t,he most prolific sources of error in measuring the pH of COz- containing solutions lie in the preparation and maintainence of the CO,-I-I, mixt,ures.

A priori, several theoretical objections presented themselves to the use of the electrode for blood or serum. First, the quinhydrone electrode functions best in an acid medium, but since blood and serum are never more alkaline than pH 8 the error introduced by this factor is not important. Our experience with standard phosphate solutions is in agreement with this conclusion.

A second difficulty, that of salt. effect, has been carefully studied by Siirensen, Sdrensen, and Linderstrijm-Lang (3) and by Biil- mann and Lund (4). In the salt concentration of blood, about 0.15 X, this effect is also negligible.

Loss of COz must be prevented, but this can be accomplished more easily with the quinhydrone electrode than wit.h the hydro- gen electrode (see below).

The possibility of error which is of most moment, is that since

* Aided by a grant from The Rockefeller Foundat,ion. i \-anderbilt Isnirersiry.

727

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728 Quinhydrone Electrode

the quinhydront electrode is an oxidation-redo&ion system, whose potential is influenced l)y pH, ot,her oxidat.ion-reduction systems in the solution being investigated may change the ratio of quinone to hydroquinone to a value different, from unity. The blood serum, tissue, and tissue fluids, contain complex systems of oxidizing and reducing enzymes, and it is to be expected that they will affect the added system. The velocity with which this ocCurs, however, may be so slow that it, is possible to deter- mine t,he in,itinl potential rlue to the pH. That this is possible for serum is shown below.

131ood itself contains such a complicated system in t.he oxy- hemoglobin-hemoglobin that it would appear almost impossible to use the quinhydrone electrode on it,. This conclusion is in agreement with our results.’

The c0nt.rol t.echniquc for pH determination with the hydrogen electrode was the same as t,hat described before by Cullen (6), with a Clark electrode as modified by (Mullen (7). COZ-HZ mixture and Ha,sselbalch refill technique were used in serum equilibmtion with known COZ tension.

E.M.F. readings were made with a 1,eeds a.nd Nort,hrup poten- tiometer, lamp, and scale galvanometcr. Readings were made to about 0.2 millivolts. For st,anclardizing the system we therefore used the arrangement described by Cullen (6) in a previous paper, measuring first the E.M.F. of the chain

Pt ! II2, A solution ’ ssturat,ed KC1 solut,ion saturated calomel electrode

with a known pH of the A solution. Using the convention that the IIg of the calomel electrode is

positive to the Pt, of the hydrogen electrode, we have

E M.P. - c,, J’II = __~

0.0001984 T

from which elI may bc calculated, and with this value of elf the same expression may be used for the calculation of pH in an

1 Our results Lvith blood are not in agreement. with those of Corran and J.ewis (5), ~vho report, normal values for normal blood obtained with the quinhydrone elcctrotle. They did not compare their determinations with those by any other method. We are not, able to explain this difference be- tween their and our experienre.

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G. E. Cullen and E. Biilmann 729

X solution by measuring the E.M.P. of the chain with S solution in place of A solution thus

1% / IIz, X solution saturated KCI solution saturated calomcl electrode I

In the same way by measuring the E.M.F. of the chain

I q~~inhydrone 1 4

“U / A solution i saturated KC1 soiution saturated calomel electrode

the corresponding value e4 for this type of chain was determined and, then, by measuring the E.M.F. of the chain

saturated I\U , quinhydrone X solution j IiCl so,ution

saturated calomel electrode

t.he pli of the X solution was calculated from

pH = E.M.P. + Cq

0.0001084 2’

In every C&SC each system was standardized against the same standard solution and the values of eH and e, determined for the whole system. In consequence, the accuracy of the measure- ments depends on t’he exactness of the pH value of the A solution but is independent of the comparison electrode (in this case saturated calomcl electrode), the liquid junction potentials, temperature coefficient, etc. The A solut,ion in our experi- ments was a st,andard phosphate, with pH close to the pII of the X solution, which was in turn checked against standard HCl solution. The Lype of comparison electrode is incidental. (When using quinhydrone electrodes and set’ting up apparatus for that purpose, the quinhydrone standard electrode may be convenient (Veibel (8) .)

Electrodes.-The large electrode vessels first used by Biilmann are not suitable for CO2 solutions and also require too much material. The capillary electrode described by Biilmann and Lund was slightly modified for our purpose. We tried four dif- ferent types of capillary electrodes.

Biilmann and Lund’s capillary electrode (Fig. 1, A) is mechani- cally more convenient in form R, because it is more rigid in its connection and thus does not draw KC1 up into the capillary as

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730 Quinhydrone Electrode

A sometimes does. The wire (gold-plated platinum wire) is shorter and better protected. The capillary part of the tube should be at least 20 mm. long and about 1 mm. inside diameter.

C is B with a coating of De Khotinsky cement over the upper half of the wire. This is used in CO, solution so that the change of pH due to loss of COz at the surface cannot affect the E.M.F. De Khotinsky cement, soft, is better than glass, because it is less fragile and can easily be removed or renewed in an alcohol flame.

A

D, made of heavy-walled capillary glass and fitt.ed with a rub- ber bulb, is the most convenient form to manipulate. It is similar to the hydrogen electrode described by Walpole (9). The sur- face of the solution is well above the electrode wire. It has, however, one drawback in that it does not immediately attain the temperature of the saturated KC1 bridge solution.

B and C are for this reason most satisfactory for serum. Two or more of these electrodes may be placed in the same KC1

bridge and by means of a convenient throw switch can be read at 15 second intervals. In order not to disturb the electrodes it is

B

1 s 3

i

FIG C

1. D

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G. E. Cullen and E. Biilmann 731

convenient to use light spring clips with serrated jaws soldered to flexible insulated copper wire as leads from the switch. We use the clips sold by Dennison for’ show-window cards. Biilmann has previously stated that gold or gold-plated electrodes may in some cases be more satisfactory than platinum, and this is also true in serum work. Because the platinum is gold-plated great care must bc taken to guard it from mercury. This is done either by using platinum wire for the whole distance or if, for economy a mercury contact is used, a copper wire should be cemented in. Cotton wool soaked in hot paraEi.n and packed in around the copper wire is most satisfactory.

The gold-plating is best applied with a very low current (some few milliamperes) from a gold cyanide solution. The electrodes may be cleaned by immersion for several hours in concentrated sulfuric acid, arsenic-free, containing chromic acid.

The quinhydrone was prepared in the following manner: 100 gm. of ferric ammonium alum are dissolved in 300 cc. of water at about 65” and this solution is poured into a warm solu- tion of 25 gm. of hydroquinone in 100 cc. of water. The quin- hydronc precipitates in fine dark needles. (If a more dilute solution of hydroquinone is used, the crystals formed by the precipitation are bigger and do not dissolve so rapidly in water.) The mixture is cooled in ice and filtered by suction, and the pre- cipitate then washed four or five times with cold wat.er and dryed in the air at room temperature. Yield 15 to 16 gm.

Before comparing the hydrogen and quinhydrone electrodes directly we’ attempted in numerous experiments to establish conditions under which we could obtain reproducible readings with the quinhydrone electrode in serum or blood. Since this was difficult and since changes in several details of technique cause error we will record our difficulties for the benefit of others.

Serum. Use of Mineral Oil to Prewnt Loss of C&.--In ob- taining strum from blood wit’hout loss of CO, it is necessary to USC a cover of paraffin oil. If one has a small amount of a COr- containing solution, such as serum, covered with oil, it is difficult to add quinhydrone, for the cryst,als remain in the oil and dis- solve in it. By first placing a generous quantity of crystals

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732 Quinhydrone Electrode

in the bottom of a dry tube, then adding oil, one can run in the solution under the oil and by stirring with a rod dissolve some quinhydrone in a minute or two. But as shown later, this re- quires too much time for serum. Next we tried moistening first the quinhydrone with a small drop of water, then adding oil, and finally the iolution. Sufficient quinhydrone was dis- solved more quickly. The plan was to make many readings on one sample, but we were unable in this way to get satisfactory successive duplicate readings on serum.

Time E$ect.-When quinhydrone is added to serum, the solu- tion begins to change color in the first 15 seconds to 1 minute and in 2 or 3 minutes a definite red color develops. It is necessary to make the reading before this color develops. Addition of sodium fluoride to the serum does not delay this color change.

Quinhydrone Added in Capillary Electrodes.-It is possible to add the quinhydrone to the capillary electrode before addition of serum, in either of two ways: (a) Dry the electrode, type C, and shake crystals of quinhydrone into the capillary portion. Then dip the capillary point of the electrode quickly through the oil, and draw up the solution by manipulation of the inside tube. (6) Touch the gilded wire, moistened with either water or 0.9 per cent NaCl solution, to powdered quinhydrone and insert the wire carrying its load of crystals into the capillary. The latter method is the more convenient and the dilution is apparently negligible. However, the upper surface of the solution is exposed to air and can lose COz. An attempt was made to use technique (a) above, adding dry quinhydrone, then drawing up a little oil first, but the oil clings to the capillary walls and often insulates the electrode and also interferes with the solution of the quinhydrone.

The effect of the COz loss may be eliminated by using an electrode of type C or D. Apparently the CO2 loss is slight and irregular, for sometimes one cannot detect any difference when this precaution is omitted, although usually one can.

ReproducimXty of Results with Capillary Electrode.--In reading the potential of serum the time of contact of serum with quinhy- drone is controlled thus: The electrode and wire are carefully rinsed with distilled water and the water blown from the capil- lary. The wet wire is then touched to dry quinhydrone and

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G. E. Cullen and E. IMmann 733

inserted into the capillary which is thon plunged through the layer of oil overlying the strum in its tube. TVithdrawing the inner tube, strum is drawn up into the capillary to above the lower end of the cement covering. The lower end of the wire should bc at least 5 mm. from the tip of t,hc capillary electrode tube. The quinhydronc is partially dissolved as the serum flows up. The time is noted most easily by starting a stop-watch,

e Zechde

lira. 2

and the electrode is attached to the connecting wire of the poten- tiometer and then placed in the saturated KC1 bridge (Fig. 2).

The first reading is finished by the half minute, then further readings are made at 1, 2, and 3 minutes. One can work with two or three electrodes at the same time. The temperature of the KC1 is also read. If there is a great change between the successive readings, more than 1 t,o 2 millivolts, the datermina- tion is not used, but in most cases the drift for the first 2 minutes is low and the 3 minute and minute readings are pract,ically the

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734 Quinhydrone Electrode

same. The minut.e reading is the one used for calculation, since temperature agreement with the KC1 has been obtained by that time and drift has not become marked. Three or four determinations must be made and averaged.

Control of System and Diffusion.-The standardization of the electrode is made in the same way. When using standard solu- tion in electrodes t.hat are satisfactory, there is no drift due to diffusion for 10 or more minutes, so that the 1, 2, and 3 minute

TABLE I.

Ezample of Reproducibility of Reading with Serum.

Solution.

PO1 pH 7.0

Serum.

Ek?c- trade No. /

III IV ’

III 18 IV 18 III 18 IV 18 III 18 IV 18

Tern- E.M.F. perature.

$ min. 1 1 min. 1 2 min. 1 3 min. I Calculation.

“C.

18 18

-0.0490'-0.0490 -0.0490 -0.0490 e = 0.4529* -0.0490 -0.04901-0.0490 -0.0490

pH at 1 min. -0.0025 -0.0025-0.0025-0.0020 7.81 -0.0028-0.0025 -0.0022 -0.0020 7.81 -0.0038 -0.0038 -0.0043 7.78 -0.0040 -0:0045 -0.0055 -0.0065 Xt -0.0030 -0.0030 -0.0037 7.80 -0.0025 -0.0028 -0.0030 7.80

Averageatlmin...........................................I 7.80

*7.0 = e + E.M.F.

0.0577 ore = 7.0 X 0.0577 + 0.0490.

t Determination rejected because of drift.

readings must agree. The electrodes should be rinsed with t,he standard solution once before adding the quinhydrone. It makes no difference whether quinhydrone is added to the standard solu- tion or to the wire as described above. As we have not always been able to obtain agreement closer than 0.5 millivolt between successive determinat,ions we always take the average of at least two and usually four determinations. We always use at least two electrodes.

A sample experiment showing the agreement between successive determinations in different electrodes is given in Table I.

The following experiment, one of several, is given as an example of the results wit.11 bot.h elect.rodes on the same serum.

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G. E. Cullen and E. Biilmann 735

Experiment to Compare Hydrogen and Quinhydrone Electrodes. -About 300 cc. of blood were taken, as treatment, from the arm vein of a patient with hypertension. The blood ran, with stirring, into a bottle containing enough NaF to make the resulting concentration about 0.3 per cent. NaF was used to prevent glycolysis and consequent change in reaction (Evans (10)) during the time required for the two sets of determinations. 100 cc. of

TABLE II.

Comparison of Hydrogen Electrode and Quinhydrone Electrode on the Same

Serum.

Ph0sphat.e solution pH 7.8 at 20”.

Electrode.

Hydrogen I. “ II.

Quinhydrone III. ‘I IV.

E.M.F’. e

volts

0.7035 0.2503 0.7035 0.2503 0.000 0.4532 0.000 0.4532

Serum equilibrated at 26 mm. CO, at 20”. pH determinations made at 20”.

Electrode.

Hydrogen I. ‘I II.

Quinhydrone III. “ IV. ‘I III. ‘I IV.

E.M.P. ( + correction for CCh).

rdte 0.6856 0.6873

+ min. 1 min. 2 min.

-0.0165 -0.0165 -0.0175 -0.0170 -0.0170 -0.0185 -0.0180 -0.0175 -0.0175 -0.0175 -0.0175 -0.0185

PH’

7.50 7.52

pH at 1 min.

7.51 7.50 7.50 7.50

* A more direct method of comparing the two electrodes is to calculate the E.M.F. of the chain

Pt 1 Hz solution 1 saturatedKCIsolution + quinhydrone 1 Au

Using the average values of the quindrone and hydrogen electrodes (corrected for vapor tension to 1 atmosphere of Hz), the potential of this chain is

Phosphate solution. . . . . . . . . .0.7037 + 0 .oooo = 0.7037 volts. Serum...........................0.6871 + 0.0171 = 0.7042 “

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736 Quinhydrone Electrode

the blood were centrifuged and the serum was removed. This is called “serum” because although the blood did not clot as a whole there were many small clots, and fibrin formed and was removed with a rod after gentle stirring from the supernatant plasma over the cells.

‘Jhc serum was cquilibrat.ed in l,wo successive tonomemrs- ~- as described by Austin and coworkers (ll)-at 26 mm. of CO2 pressure at 20” and removed to a mercury sampling tube from which portions were run under oil int,o small short tubes into which it was convenient to plunge the quinhydrone capillary electrodes. ‘The results arc given in Table II. The agreement between t,he two types of electrodes is within 0.02 ~1-1, which is within the error of the two methods.

E.rpriments with Blood.

Since it’ would be of great. value to be able to use the capillary clcct,rode directly on blood, we made many at,tcmpts to work fast enough to do this. With a small amount of quinhydrone the drift is so rapid and errat.ic that we could not extrapolate back to zero time. If, however, a large amount of quinhydrone is added to blood, within 10 seconds the blood becomes dark, t.he color of methemoglobin. Gas comes off in minute bubbles and the resulting dark mixture gives a stable potential, which does not, however, correspond to the potential of the serum from the un- treat.ed blood, but to a much mom alkaline reaction. Uhough it is possible that this value may have a simple constant relation to the actual value, it seems so improbable t,hat we have not investigated the possibility further. The drift in serum is not stabilized at any new value by adding large amounts of quin- hpdrone.

Serum Containing IIemoglobin.- Serum containing definite traces of hemoglobin gives drifting values and is, therefore, unsuitable for study with the quinhydrone electrode.

Serum and Plasma.--The reproducibility of the results with successive determinations illustrated for serum in Table I holds also for plasma. However, all our comparison experiments with both the hydrogen and quinhydrone electrodes were done on serum from blood containing Sal’, as described above. Although this may have contained some fibrin we cannot now state that

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G. E. Cullen and E. Riilmann 737

plasma can be used with as much satisfaction as serum. It is, however, probable that it can be. This question Cl1 be reported on later.

COSCLUSIONS.

Our experiments with the solutions used in t’his study furnish further evidence of the convenience of the quinhydronc electrode wherever secondary oxidation-reduction reactions do not inter- fere. The capillary electrode .is an economical and convenient form of the electrode. There is: however, t’hc same tendency for individual electrodes to vary as with the hydrogen electrodes, so that the safest procedure is to check the accuracy of the entire system against a suitable st.andard solution. The immediate standard solution used should preferably be one with pH near the pI1 of the unknown solutions.

In biological material, it, is essential to determine whether the enzymatic processes interfere and, if so, whether the rate of such effect is sufficiently slow to allow determinations.

Serum.-In the case of scruq proper precautions as to time make it possible to use the quinhydrone electrode with apparently the same accuracy as the hydrogen electrode.

Blood.---Our results with Iv-hole blood indicat,e that the quin- hydrone electrode is not suit.able for use with the hemoglobin system.

We wish t,o thank Prof. C. Lundsgaard for putting his labora- tory at t,he Rigshospitalet at, our disposal.

The use of the quinhydrone electrode for determining the PI-I of blood and blood serum has been studied.

By reading the potential immediately after addition of quin- hydrone satisfactory results arc obtained with serum. The de- tails of a technique for this purpose are described.

We have not been able to use the quinhgdrone electrode with whole blood.

BIBLIOGHAPIIT.

1. Biilmann, IX., Ann. chim., 1021, xv, series 9, 109 2. Biilmnnn, IS:., ‘I’?. Fnrcrtlny Ser., 1923, six, 67G.

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Quinhydrone Electrode

3. Serensen, S. P. L., SGrensen, M., and Linderstrijm-Lang, Ann. chim., 1921, xvi, series 9,283.

4. Biilmann, E., andLund, H., Ann. chim., 1921, xvi, series9,321. 5. Corran, J. R., andLewis, W. C. hlcC., Biochem. J., 1924, xviii, 1358. 6. Cullen, G. E., J. Biol. Chem., 1922, lii, 501. 7. Cullen, G. E., J. Biol. Chem., 1922, lii, 521. 8. Veibcl, S., J. Chem. Xoc., 1923, cxxiii, 2203. 9. Walpole, G. S., Biochem. J., 1914, viii, 131.

10. Evans, C. L., J. Physiol., 1922, lvi, 146. 11. Austin, J. IT., Cullen, G. E., Hastings, A. B., McLean, F. C., Peters,

J. I’., and Van Slyke, D. D., J. BioZ. Chem., 1922, liv, 121. i’2. Clark, \V. M., The determination of hydrogen ions, Baltimore, 2nd

edition, 1922.

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Glenn E. Cullen and Einar BiilmannON SERUM

CONCENTRATION DETERMINATIONELECTRODE FOR HYDRION

THE USE OF THE QUINHYDRONE

1925, 64:727-738.J. Biol. Chem. 

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