TITRIMETRIC DOUBLE HYDROGEN OR QUINHYDRONE ELECTRODE SYSTEMS FOR HYDRION DETERMINA-

TION; APPLICATIONS TO URINE AND BLOOD.

BY GEORGE H. MEEKER AND BERNARD L. OSER.

(From the Laboratories of Biochemistry of the Philadelphia General Hospital and the Graduate School of Medicine of the University

of Pennsylvania, Philadelphia.)

(Received for publication, October 22, 1925.)

In this paper are presented the details of a sirnplified method for determining hydrion concentration electrometrically, requiring only the use of electrodes and a galvanometer, but dispensing with the potentiometer and the standard cell.

Klopsteg (1, 2), in 1920, suggested the use of the double hydro- gen electrode in electrometric titrations. The apparatus he used consisted of two hydrogen electrodes, one immersed in a buffer solution of known pH, the other in the solution to be titrated to that pH. A galvanometer with protective resistance and a tapping key completed the circuit. Klopsteg specifically stated, however, that the method “can be used only for titrating and that the titration can be carried only to the end-point which is deter- mined by the standard solution” and that for measurement of pH a potentiometer must be used. Our purpose is to demon- strate the applicability of the same arrangement for the measure- ment of pH.

In the course of the experimental work on the double hydrogen electrode system it was realized that the step in the manipulative process that required special care was the preparation of Ohe electrodes themselves, and that this factor could be eliminated by the use of quinhydrone electrodes. This permits the use of un- platinized metallic electrodes, and dispenses with hydrogen generation, bubbling, etc. The quinhydrone system thus has the further advantage of being adaptable to the measurement of the

307

by guest on April 3, 2019

http://ww

w.jbc.org/

Dow

nloaded from

308 Titrimetric Determination of pH

pH of C&containing fluids such as are met with in biological work.

Briefly, t,he method consists in balancing a potential set up in a half-cell containing a solution of unknown pH, with that produced by a known mixture of two solutions of a buffer pair in the other half-cell, the proportions of the latter constituting a measure of the pH of the unknown solution, inasmuch as at zero deflection of the galvanometer the hydrogen ion concentrations of the solu- tions in the opposing half-cells are the same.

Apparatus.-For double hydrogen electrode work any con- venient type of bubbling electrode, such as described in Clark’s book (3), could be used. In the present experiments the Hilde- brand electrode was modified to increase its stability, thus eliminating electrode supports. The jacket was shorter and had a conical bell. The electrodes t,hemselves were platinized in the customary manner (Clark), rotating slowly to insure uniformity of the deposit.

The electrodes used in the quinhydrone system consisted simply of heavy platinum or 24 carat gold wire sealed through glass tubes about 10 cm. long and 2 mm. internal diameter. The gold wires were sufficiently long to permit direct contact with the copper leads, while in the platinum electrodes contact was made through mercury.

Saturated potassium chloride was used in the salt bridge. Hydrogen was delivered from a cylinder and passed through alkaline pyrogallol and distilled water.

An inexpensive galvanometer (Leeds and Northrup potentiom- eter galvanometer No. 2320d) was found to be sufficiently sensi- tive for this work. In fact the sensitivity of the galvanometer recommended for t,he type K potent.iometer equipment exceeds that of the titration process and is therefore less desirable. Addi- tional resistance in the circuit is not really necessary from the point of view of protection to the galvanometer. If the stops in the instrument are so adjusted as to confine the deflection of the pointer within scale limits such resistance may be dispensed with entirely. A tapping key was also introduced into the circuit.

Procedure for the Double Hydrogen Electrode System.-The solution whose pH was to be measured was introduced into one of the half-cells (reference cell) and a measured volume of one of the

by guest on April 3, 2019

http://ww

w.jbc.org/

Dow

nloaded from

G. H. Meeker and B. L. Oser 309

buffer pair1 into the other. The hydrogen electrodes were im- mersed in the solutions and connected with the hydrogen supply. Hydrogen was allowed to bubble through the solutions sufficiently to saturate them as well as the electrodes. The time necessary for this was previously determined as complete saturation is of funda- mental importance. 15 minutes were allowed for this step, and both solutions were kept saturated with hydrogen throughout the procedure. The salt bridge was then introduced and the other solution of the buffer pair delivered into the titration cell from a burette. The key was tapped at intervals, more frequently as the extent of the deflection of the galvanometer diminished. At zero deflection a reading of the burette was made. The electrode from the reference cell was then immersed in the buffer solution and the equality of the electrodes confirmed by producing zero deflection of the galvanometer. Following this the electrode was returned to its original solution and another drop or two of buffer added to produce deflection in the opposite direction.

The necessity for equivalence of the two hydrogen electrodes cannot be emphasized too strongly. They should be platinized in exactly the same manner, completely saturated with hydrogen, and checked for parity by zero deflection when immersed together in one cell, before the titration is begun.

The volumes of the buffer pair, the pH of whose mixture equals that of the unknown solution, were now known. Curves were constructed from the data for each buffer system (the most im- portant of which are found in Clark (3)) in which the proportion of one of the solutions in the final mixture was plotted against the pH. From these curves was read directly the pH of the unknown solution. Examples of such curves are given in Fig. 1.

In Table I are given the results obtained by using as “unknown” solutions, standard buffer solutions of definite pH, and balancing against them the buffer solutions as indicated above. The final values were read from the curves in Fig. 1.

1 The selection of the buffer solution depends, of course, on the range of pH in which the unknown solution falls. This can be readily estimated by the use of a set of indicators such as those of Clark and Lubs. For in- stance, if a solution is “alkaline” to methyl red and “acid” to phenol red, its pH must fall somewhere between the ranges of these two indicators, namely pH 6 to pH 7. In this case buffer solutions are selected cover- ing this range. If the indicators of Clark and Lubs are used, the color chart in Clark’s book will be found helpful.

by guest on April 3, 2019

http://ww

w.jbc.org/

Dow

nloaded from

310 Titrimetric Determination of pH

Procedure for the Quinhydrone Electrode System.-The first practical application of the quinhydrone electrode to the deter- mination of pH was due to Biilmann (4). The theoretical con- siderations upon which he based its use were founded on the conception of t,he electrode being in effect a hydrogen electrode at low partial pressure of hydrogen. Recently the electrochemical view-point has received emphasis (3, 5) in which the oxidation-

FIG. 1.

A, Walpole’s acetate mixtures (per cent sodium acetate). B, McIlvaine’s phosphate-citric acid mixtures (per cent disodium

phosphate). C, Stirensen’s phosphate mixtures (per cent disodium phosphate). D, Palitsch’s borax-boric acid mixtures (per cent borax).

reduction potential is attribut.ed to an effective electron pressure, resulting from the equilibrium

CGHd(OH)z = CeH402 + 2H+ + 20.

In the det,erminations here report.ed it should be emphasized that pot,entials were not measured but were simply balanced.

Quinhydrone was prepared from hydroquinone by the act’ion of ferric alum, following the method of Biilmann and Lund (6).

by guest on April 3, 2019

http://ww

w.jbc.org/

Dow

nloaded from

G. H. Meeker and B. L. Oser 311

The trace of iron salt remaining was found not to interfere with the determinations.

The unknown solution and the measured volume of one solution of the buffer pair were introduced into their respective beakers, and just preliminary to establishing the connection with the salt

PH

4.03 4.25 4.60 4.98 5.90 6.00 6.50 7.00 7.10 7.20 7.30 7.50 7.65 8.10 8.15 9.00

0.1 N HOAc 0.1 “ “ 0.1 “ NaOAc 0.1 “ HOAc M/15 KH,POa M/15 cL M/15 “ M/15 “ M/5 KH,PO, Universal buffer. M/15 KH,POd Universal buffer. M/15 KH,POh H3B03-NaCl

“

cc. cc.

8.0 0.1 N NaOAc 2.03 10.0 0.1 “ “ 4.05 10.0 0.1 I‘ HOAc 10.40

3.0 0.1 “ NaOAc 7.02 20.0 ~/15 Na2HP04 2.36 10.0 M/15 “ 1.39 10.0 M/15 “ 4.80 10.0 M/15 “ 16.10

5.0 ~/5 NaOH 3.30 t 10.0 M/lo “ 10.00

7.0 ~/15 Na2HP04 23.60 t 10.0 ~/5 NaOH 5.75

5.0 ~/15 Na2HP04 35.50 10.0 ~/20 Na2B40T 4.25 10.0 M/20 “ 5.00 10.0 H3B03-NaCl 2.60

T

TABLE I.

Double Hydrogen Electrode System.

Titration cell.

Buffer A. Buffer B.

: i

-

-

*. .,o 2 - Per cent 10.: 28.2 L9.C '0.C 10.1 12.: s2.1 il.: 19.2

'7.1

<7.: !9.2 13.: '9.1

a 2

2 x a -

.0:

.2E

.6C

.9E

.92

.Ol

.51

.Ol

.ia

.2c

.31

.52

.64

.OE

.16

.9E

d s E r-2 n

-0.01 -0.01

0.00 -0.02 -0.02 -0.01 -0.01 -0.01

0.00 0.00

-0.01 -0.02 -0.01 -0.02 -0.01 -0.02

* Ratio, in per cent, of the more basic solution of the buffer pair in the final mixture (Buffer A plus Buffer B).

t The universal buffer solution of Acree and associates, which covers the range pH 1 to 12.

bridge an excess (enough to cover the tip of a spatula) of quin- hydrone was added to each solution and stirred thoroughly for about a minute. Since quinhydrone is rather insoluble equilib- rium is reached quite readily. The second solution of the buffer pair was then added from a burette as in the foregoing method, the mixture being stirred constantly. The burette reading at zero

by guest on April 3, 2019

http://ww

w.jbc.org/

Dow

nloaded from

312 Titrimetric Determination of pH

deflection was noted and an additional drop or two of solution in- troduced, whereupon deflection occurred in the opposite direction.

It was noted that, other conditions being the same, greater deflections were produced with platinum electrodes than with gold. Since degree of deflection is the criterion of the method, platinum should therefore be preferred, provided it is inert to the particular system being measured. In some cases, such as in whole blood, the platinum behaves as an oxidative catalyst, thus rendering necessary the use of gold, which is inert in this respect (7).

TABLE II.

Double Quinhydrone Electrode System. __

PH

7.00 7.05 7.40 7.72 7.75 7.90 8.04 8.05 8.16 8.50

-

, -

Titration cell.

Buffer A.

cc.

M/15 NazHPO* 10 M/15 “ 15 M/15 “ 10 M/15 “ 26. Ml15 “ 10 M/15 “ 25. M/15 “ 18. M/15 “ 25. H3B03-NaCl 10.

“ 10.

-

-.

5

0 9 0 0 0

--

Buffer B.

cc.

M/15 KHzP04 6.8 M/15 “ 7.75 Ma/15 “ 3.9 M/15 ic 3.0 M/15 “ 1.00 M/15 “ 1.8 M/15 “ 1.0 Ml15 LL 1.2 M/20 Na2B407 5.15 M/20 cl 10.55

2

-

,er cm 59.6 66.0 71.9 89.8 91.9 93.3 95.0 95.4 34.0 51.8

6.98 7.07 7.40 7.71 7.78 7.92 8.02 8.07 8.19 8.53

m: B D 9 n

-0.02 to.02

0.00 -0.01 to.03 to.02 -0.02 to.02 to.03 +0.03

The final calculation and readings of pH were made by reference to the same curves as described above. Examples of pH titrations using the quinhydrone system are given in Table II.

The chief limitation to the application of the quinydrone elec- trode system rests on its instability in alkaline solution. La Mer and Parsons (5) investigated the relative effects of autoxidation, ionization, and the acid behavior of hydroquinone in alkaline solution by comparing measurements with the hydrogen and quinhydrone electrodes in potentiometric titrations. These authors concluded that pH 8.0 represented the maximum alkalinity

by guest on April 3, 2019

http://ww

w.jbc.org/

Dow

nloaded from

G. H. Meeker and B. L. Oser 313

for which the quinhydrone electrode system would be applicable. Schaefer and Schmidt (8) have more recently made a comparative study of the two methods and suggest pH 9.0 as the limit of alkalinity.

Inasmuch as it was our ultimate object to apply the quinhydrone method to blood, preliminary titrations were made with buffer solutions covering the range of pH 7.0 to pH 8.0. These results are tabulated in Table II, and indicate that measurements may be made within this range with reliance. It was observed however that if too much time elapsed during the titration, erroneous results were produced. From 2 to 3 minutes was generally the duration of a complete determination from the time of addition of quinhydrone until the end-point was reached.

The altered activities of quinone and hydroquinone in solutions of high salt concentration preclude the usefulness of the quinhy- drone system in such media. Sorensen, Sorensen, and Linder- Strom-Lang (9) have investigated this phase of the problem and have found that it is not appreciable in concentra6ions lower than ~/5. This factor must be taken into account in the practical application of the procedure to physiologic fluids.

DISCUSSION.

The agreement, it will be seen, is quite close in both electrode systems. The sensitiveness of the method is, of course, dependent not only on the size of the drop delivered from the burette, but also on the slope of the titration curve at the particular range involved. The latter may however be increased where greater sensitiveness is desired by extending the coordinates along the pH axis, but this obviously should not be carried beyond the limit of sensitivity of the other factors.

The buffer solutions tabulated in the text-books of Clark (3) or Michaelis (10) have been standardized potentiometrically, and are entirely dependable, provided, of course, the purity of the salts conforms to the standards set by the various investigators. If it is desirable to use as few solutions as possible to cover a wide range of pH, buffer pairs like those of McIlvaine (11) (see Fig. 1) or Acree and associates (12) may be used.

by guest on April 3, 2019

http://ww

w.jbc.org/

Dow

nloaded from

314 Titrimetric Determination of pH

Applications of the Quinhydrone Electrode to the Determination of pH.

Urine.-Schaefer and Schmidt (8) have reported a long series of experiments on the pH of urine determined potentiometrically, using on the one hand the hydrogen electrode, and on the other the quinhydrone electrode. In the latter case the reference electrode also consisted of a quinhydrone electrode, the solution being 0.1 N HCI-KCI, in the proportion of 1 of the acid to 9 of the salt,. The data of t,hese authors also include calorimetric estimations, and all these methods gave concordant results.

In the experiments here reported, calorimetric determinat,ions of the pH of urine were made at dilutions of 1: 20, using Clark and Lubs indicators. Walpole acetate mixtures and Sorensen phos-

TABLE III.

Comparative pH Determinations of Urine.

Calorimetric method. Quinhydrone method.

6.8-7.0 6.98 6.5 6.53 5.2 5.22 6.6 6.64 5.4 5.47

6.8-7.0 6.86 6.6-6.8 6.72

phate mixtures constituted the standard buffer solutions, and these varied by intervals of 0.2 pH.

In order to eliminate the subjective error, where the pH of a specimen lay between two standards, the pH values of the latter were recorded instead of an estimated value.

The electrometric titrations were conducted on urine diluted approximately 1: 20, following the technique outlined above. Typical results are presented in Table III.

This method is useful in measuring hydrion concentration in cases where the solutions are too deeply colored or turbid, as well as in those cases (e.g., certain urines) where the dichromatic effects of the indicator render calorimetric comparison difficult.

Blood.-Success had been claimed for the quinhydrone electrode in the potentiometric estimation of the pH of blood by Corran

by guest on April 3, 2019

http://ww

w.jbc.org/

Dow

nloaded from

G. H. Meeker and B. L. Oser 315

and Lewis (13), a calomel cell serving as the reference electrode. Since saturation with quinhydrone without loss of CO2 was a necessary step, their use of undiluted whole blood appears to be an objectionable feature. Similar criticism might be applied to the syringe electrode described by Mislowitaer (14) for pH measurements of blood.

The experiments of Corran and Lewis were not confirmed by Cullen and Biilmann (15). These authors describe a capillary quinhydrone electrode for estimating the pH of undiluted plasma or serum by means of the potentiometer. Their experience with whole blood however was unsatisfactory.

The titrimetric procedure adopted for the determination of the pH of blood is as follows: The reference cell for the blood was prepared with 10 cc. of physiological saline solution to which was added an excess of quinhydrone. After vigorous stirring, a layer of neutral mineral oil was added and the beaker immersed in a bath at 38°C. A thermometer was suspended in this reference cell and a gold electrode, which had already been connected to the rest of the circuit, was immersed. When the temperature of the contents of this cell reached 38”, there were added to the titration cell 10 cc. of ~/15 disodium hydrogen phosphate and an excess of quin- hydrone, and the other gold electrode was then immersed in it. 0.8 cc. of oxalated whole blood, collected without loss of COZ, was then introduced into the saline un.der oil and stirred thoroughly for 20 to 30 seconds. The salt bridge connection was then made and the Gtration with ~/15 potassium dihydrogen phosphate con- ducted without delay, using a micro burette. During this short space of time the temperature of the diluted blood was maintained at 38” without difficulty. The final pH values were obtained as previously indicated from the Sorensen phosphate curves.

While no altogether satisfactory method for the calorimetric determination of the hydrogen ion concentration of blood exists, that of Cullen (16) makes the closest approach to it. The technical simplification introduced by Hawkins (17) permits the use of small quantities of blood. The only uncertain factor in the method is the empirical correction that must be applied to bring the pH as determined calorimetrically at room temperature lo the value obt,ained electrometrically at 38°C. according to the equation

pH3B0 = pHtO + 0.01 (t - 20) - 0.23

by guest on April 3, 2019

http://ww

w.jbc.org/

Dow

nloaded from

316 Titrimetric Determination of pH

According to Hastings and Sendroy (18) no correction is neces- sary if the reading is made with both the standard and the diluted plasma at 38°C. This has been confirmed by Drucker and Cullen (19) but in agreement with these authors we find it more con- venient to work at room temperature and apply the correction, which for human blood appears to be quite constant (-0.23 =!z 0.04). The original Cullen standards were used, inasmuch as no greater permanence was observed with the bicolor standards of Hastings and Sendroy.

In Table IV are recorded the results by the two methods of some determinations conducted on the blood of hospital patients. Considering the slight individual variations in the Cullen correc- tion the agreement between the two methods is satisfactory.

TABLE IV.

Comparative pH Determinations of Blood.

Calorimetric method. Quinhydrone method.

7.37 7.41 7.42 7.39 7.41 7.45 7.63 7.62 7.42 7.38 7.36 7.34

SUMMARY.

Difference.

+0.04 -0.03 +0.04 -0.01 -0.04 -0.02

1. A simplified electrometric method for the measurement of hydrogen ion concentration is presented based on the use of the double hydrogen electrode or the double quinhydrone electrode, in which the potential of the measured solution in one half-cell is balanced by titrating standard buffer solutions into the other half- cell. The proportions of the latter solutions required to produce equilibrium determine the pH of the unknown solution.

2. Applications of the quinhydrone electrode to the estimation of the pH of urine and blood are described, which give results concordant with the calorimetric methods now in general use.

The authors wish to express their indebtedness to Dr. D. Wright Wilson and to Dr. Glenn E. Cullen for their helpful criticism in the preparation of this paper.

by guest on April 3, 2019

http://ww

w.jbc.org/

Dow

nloaded from

G. H. Meeker and B. L. Oser 317

BIBLIOGRAPHY.

1. Klopsteg, P. E., Science, 1920, Iii, 18. 2. Klopsteg, P. E., Ind. and Eng. Chem., 1922, xiv, 399. 3. Clark, W. M., The determination of hydrogen ions, Baltimore, 2nd

edition, 1923. 4. Biilmann, E., Ann. chim., 1921, series 9, 109. xv, 5. La Mer, V. K., and Parsons, T. R., J. Biol. Chem., 1923, lvii, 613. 6. Biilmann, E., and Lund, H., Ann. chim., 1921, xvi, series 9, 321. 7. Dixon, M., and Quastel, J. H., J. Chem. Sot., 1923, cxxiii, 2943. 8. Schaefer, R., and Schmidt, F., Siochem. Z., 1925, clvi, 63. 9. SGrensen, S. P. L., Siirensen, M., andLinderstrBm-Lang, K., Ann. chim.,

1921, xvi, series 9, 283. 10. Michaelis, L., Die Wasserstoffionenkonzentration, Berlin, 2nd edition,

1923. 11. McIlvaine, T. C., J. Biol. Chem., 1921, xlix, 183. 12. Acree, S. F., Mellon, R. R., Avery, P. M., and Slagle, E. A., J. Infect.

Dis., 1921, xxix, 7. 13. Corran, J. W., and Lewis, W. C. McC., Biochem. J., 1924, xviii, 1358. 14. Mislowitzer, E., Biochem. Z., 1925, clix, 77. 15. Cullen, G. E., and Biilmann, E., J. Biol. Chem., 1925, Ixiv, 727. 16. Cullen, G. E., J. Biol. Chem., 1922, iii, 501. 17. Hawkins, J. A., J. Biol. Chem., 1923, lvii, 493. 18. Hastings, A. B., and Sendroy, J., Jr., J. Biol. Chem., 1924, Ixi, 695. 19. Drucker, P., and Cullen, G. E., J. BioZ. Chem., 1925, lxiv, 221. by guest on A

pril 3, 2019http://w

ww

.jbc.org/D

ownloaded from

George H. Meeker and Bernard L. OserURINE AND BLOOD

DETERMINATION; APPLICATIONS TO SYSTEMS FOR HYDRION

OR QUINHYDRONE ELECTRODE TITRIMETRIC DOUBLE HYDROGEN

1926, 67:307-317.J. Biol. Chem. 

  http://www.jbc.org/content/67/1/307.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  ml#ref-list-1

http://www.jbc.org/content/67/1/307.citation.full.htaccessed free atThis article cites 0 references, 0 of which can be by guest on A

pril 3, 2019http://w

ww

.jbc.org/D

ownloaded from