A MICROQUINHYDRONE ELECTRODE : ITS APPLICATION TO THE DETERMINATION OF THE pH OF

GLOMERULAR URINE OF NECTURUS”

BY J. A. PIERCE AND HUGH MONTGOMERY

(From the Harriet Lane Home for Invalid Children, the Johns Hopkins Hospital, Baltimore, and the Laboratory of Pharmacology, University of

Pennsylvania, Philadelphia)

(Received for publication, June 8, 1935)

The problem of determining the pH of rachitic cartilage was suggested to one of us (Pierce) by Dr. E. A. Park of the Johns Hopkins Hospital. It seemed possible that a small quinhydrone electrode could be constructed for direct insertion into the tissue and thus be used to measure its reaction in situ. The first at- tempts showed that accurate pH measurements of known buffer solutions could be made with an electrode consisting of a 36 gage platinum wire (0.08 mm. in diameter), the end of which was coated with quinhydrone crystals. It became obvious, however, that an unprotected platinum wire was unsuitable for direct inser- tion into tissues. Accordingly, we adopted the suggestion by Dr. Richards of enclosing it in the quartz capillary pipette described by Wearn and Richards and used by them in collecting fluid from the renal corpuscle of frogs (1). The result of the work which followed is the apparatus described below. Consistently reliable measurements of the pH of fractions of a c.mm. of fluid have been made with it. It resembles the capillary electrodes of Cullen and Biilmann (2) and Cullen (3) more closely than any others which we have seen described in the literature. It differs, however, in that it requires far less fluid for a determination and provides complete prevention of loss of COz.

* This work was financed in large part by a grant to the University of Pennsylvania from the Commonwealth Fund of New York and by the John Howland Memorial Fund of the Johns Hopkins University. The earliest stages of the work were conducted in Baltimore; the apparatus was developed and most of the experiments here described were made in Philadelphia.

763

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764 Microquinhydrone Electrode

In developing the technique of its application to biological sub- stances it seemed wise to choose familiar material. Data had recently been obtained which showed that the pH of glomerular urine from frogs and Necturi can be measured with accuracy by microcolorimetry, and is the same as that of plasma (4). It was decided to postpone the study of cartilage and to work first with glomerular urine in the large corpuscles of living Necturi. In this way flaws in technique would presumably be more easily disclosed. It was also of obvious advantage that the calorimetric work on the reaction of glomerular urine should be controlled by measurements by another method.

Microcapillary Electrode-The apparatus of which the micro- electrode is the essential part is of the same design as that origi-

4

FIG. 1. fiilicrocapillary electrode. A, B, and C, successive stages in assembling; 1, quinhydrone-encrusted end of wire; 8, de Khotinsky cement on wire; 3, ring of de Khotinsky cement on large end of pipette; 4, end of long arm of a-way stop-cock tube.

nally described by Wearn and Richards (1). The pipette which contains the electrode is made from transparent quartz tubing not less than 0.5 mm. in internal diameter; its length is about 5 cm. Care should be taken that the taper of the point be blunt “‘and symmetrical. The diameter of the point is about 10~.

A piece of 36 gage platinum-iridium wire about 10 cm. in length is held taut with forceps and passed through an alcohol flame to straighten it. It is then dipped into distilled water, removed, and allowed to dry, care being taken to prevent contamination. A saturated solution of LaMotte’s Standardized quinhydrone in redistilled acetone is prepared in a shallow dish, one end of the wire is allowed to rest in it to a depth of about 2 mm., and a current of air played on the surface. A tenacious crust of quinhydrone about 0.01 mm. thick is left on the wire (see Fig. 1). All but 1

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J. A. Pierce and H. Montgomery 765

mm. or less of the quinhydrone-encrusted end of the wire is cut off with scissors. A minute drop of de Khotinsky cement is melted onto the wire 1 to 2 cm. from the coated end, and this end of the wire is inserted into the quartz tube as far as it will go. It is important that the wire be straight. The pipette is passed laterally over the flame of a microburner; the drop of de Khotinsky cement melts and attaches the wire to the pipette without occlud- ing the lumen. The protruding end of the wire is bent sharply around the large end of the pipette and fixed in place by a thick ring of de Khotinsky cement. The pipette is filled with clean mercury with the aid of a capillary pipette and cemented into the end of the long, mercury-filled arm of the S-way stop-cock tube held in the micromanipulator.

Scrupulous cleanliness of quartz tubing and platinum-iridium wire is essential. Care must be taken not to heat the quinhydrone when melting the cement. Excessive heating of the cement is to be avoided. The electrodes, kept free from contamination, do not deteriorate quickly. We have chosen arbitrarily, however, to use none older than 24 hours. An electrode can be used for several successive determinations provided it is thoroughly rinsed with distilled water.

When the apparatus is properly assembled we have a mercury- filled system consisting of leveling bulb, rubber tubing, 3-way stop-cock tube held firmly in the micromanipulator, and ending in the quartz pipett,e which contains the electrode. The point of the pipette is inserted into the fluid to be studied, and, by manipulating the stop-cock and leveling bulb, the fluid flows into the pipette as the mercury recedes and comes into contact with the quinhydrone. 0.1 c. mm. of fluid is adequate for a determination; satisfactory results have been obtained with volumes smaller than this.

The electrode, protected by the quartz pipette and the mercury in it, is not contaminated by extraneous material through which it may have to be passed to reach the fluid to be tested. The fluid taken into the pipette does not come in contact with air.’

The quinhydrone electrode was combined with a saturated KCl-

1 It occasionally happens that a minute bubble of air, trapped in the pipette, remains in the fluid to be examined. It has not been found that its presence affects the determination.

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766 Microquinhydrone Electrode

calomel half-cell in the conventional way. The normal potentials of the saturated KCl-calomel half-cell at the respective tempera- tures were those of Michaelis (Clark (5) p. 314). The normal potentials of the quinhydrone electrode were those given by Clark ((5) p. 672). In all cases the E.M.F. of the liquid junction was assumed to be 0. Calculations were made by use of the equation

pH = &,. -&a,. - E. M. F.

0.0001982 T

where Eqh. = the normal potential of the quinhydrone electrode; E Cal. = the normal potential of the saturated KCl-calomel elec- trode; T = absolute temperature.

Potential measurements were made with a Leeds and Northrup portable potentiometer (catalogue No. 7655) from which the in- cluded galvanometer had been removed. An enclosed lamp and scale galvanomete? (Leeds and Northrup catalogue No. 2420-C) was substituted. Temperature was recorded with standardized thermometers; that of the fluid in contact with the electrode was estimated to ~tO.3”.

In control tests of the accuracy and reliability of the apparatus known buffer solutions were taken into the electrode pipette from a small beaker; the liquid junction to the calomel half-cell was established with a saturated KCl-2 per cent agar bridge. The results of these tests were compared not only with the calculated pH values of the solutions3 but also with the results of determina- tions made on the same solutions by means of a quinhydrone electrode of the usual type (14 gage platinum wire, fused into a glass tube; quinhydrone crystals added to the fluid).

Table I contains typical results obtained in twenty such com- parisons, made consecutively. Twelve microelectrodes were used, no one in more than three determinations. Values obtained at room temperature have been reduced to 20” by adding a factor (0.00155 X (t - 20)) derived from the data of Hastings and

* For increased ease of reading, the lamp of the galvanometer was so adjusted that the beam crossed the 0 line of the scale at an acute angle. This adjustment allows detection of 0.5 millivolt at the high resistance produced by the small opening of the point of the capillary pipette.

3 The solutions were prepared according to Hastings and Sendroy (6) and the pH values calculated from their data.

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J. A. Pierce and H. Montgomery 767

Sendroy (6). They show that the accuracy which we have attained with the microelectrode was of the same order as that with the macroelectrode. It is also obvious from such results that the accuracy of the determinations by the microelectrode is not affected either by the contacts which exist within the pipette between metallic platinum, mercury, and the fluid tested or by the electrical resistance which results from the small size of the orifice of the pipette. Tests for possible errors due to polarization of the electrode in these minute quantities of fluid showed that they were negligible.

TABLE I pHzo of M/i5 Phosphate Bu$ers

Calculated

7.36 7.26 7.41 7.44 7.21 7.66 7.61 7.15 7.24 7.63

Micro

Determined

MrtCl-3 .

7.37 7.25 7.40 7.42 7.22 7.67 7.63 7.16 7.22 7.60

-

7.39 7.26 7.41 7.44 7.20 7.68 7.60 7.17 7.26 7.61

-

Calculated

7.21 7.71 7.56 7.50 7.31 7.46 7.53 7.39 7.34 7.18

Determined

T MitCCO --

7.21 7.67 7.57 7.50 7.33 7.45 7.53 7.37 7.33 7.17

- Nicro

7.20 7.67 7.56 7.49 7.31 7.47 7.57 7.41 7.38 7.19

Determinations of pH of Glomerular Fluid-Adult Necturi were anesthetized by immersion in 1.5 per cent urethane for about 15 minutes; anesthesia was maintained throughout the experiment by keeping the gills in a more dilute solution. The kidneys were exposed without hemorrhage in the usual way. The holder con- sisted of a sheet of cork cemented to the bottom of a shallow rectangular glass dish t.0 which the animal was fastened by thorns.” To establish the liquid junction between the calomel cell and the quinhydrone electrode the tail, with tip cut off, was allowed to hang over the end of the holder and to dip into a beaker containing

4 To avoid extraneous sources of potential no wet metal was allowed on the holder or in contact with the animal.

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768 Microquinhydrone Electrode

saturated KC1 solution into which the end of the side arm of the calomel half-cell projected.5 The possibility of error from this device was tested directly. Two calomel half-cells were used, one in liquid junction with the glomerular urine in situ by means of a micropipette filled with Ringer’s solution, and the other in liquid junction with t,he saturated KC1 solution (into which the animal’s tail projected) by means of Ringer’s solution in agar. Control measurements were made with the point of the pipette placed in the saturated KC1 solution. In thirty-one determinations on three animals, the range of E.M.F. (deviation from control) was -0.0018 to +0.0031 volt, average +O.OOll. This corresponds to pH differences of +0.03 to -0.05, average -0.018. No consistent variations dependent upon degree of cutting of tail, or depth of immersion of the tail in the saturated KC1 solution, were found. Since the error is variable and small, values for pH of glomerular fluid and tubular fluid have not been adjusted according to these findings.

With the aid of the micromanipulator and binocular microscope the point of the electrode pipette was thrust through a capsule of Bowman so that the point was free in the intercapsular space. Temperature changes due to evaporation and possible loss of CO2 from the glomerular’ urine by diffusion into air were prevented by allowing mineral oil to drip continuously on the surface of the kidney over the glomerulus and the tip of the pipette.6

The temperature of the room varied very little during the course of an experiment. The temperature reading used in the calcula- tions was that of a thermometer in the oil which supplied the drip for the surface of the kidney. Usually within a minute after the glomerular urine reached the quinhydrone the E.M.F. readings became constant.

5 Experiments have been made which indicate that the pH of the fluid in the electrode pipette can be measured by direct insertion of the tip of the pipette into the KC1 solution which is connected with the calomel half- cell provided precautions are taken to prevent diffusion of the KC1 into the fluid in contact with the quinhydrone. The tip of the pipette may be sealed by thrusting it through a thin disk of KCl-agar orthe pipette can be prepared so that there is a constriction of the lumen between the end of the electrode and the tip of the pipette (see p. 773).

6 In many of the experiments, as an added precaution against loss of COZ, the oil had previously been equilibrated against 1.5 per cent CO* in oxygen, approximately the CO2 tension of Necturus plasma.

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J. Ai Pierce and H, Montgomery

We have applied the technique described above to glomerular fluid formed during aortic perfusion of Necturus kidneys with sodium phosphate buffer solutions of known pH. In eight experi- ments the results of microdeterminations of pH of the glomerular fluid were compared with those of macroquinhydrone deter- minations of pH of the perfusion fluid. The results are given in Table II.

TABLE II

PerJ‘usion of Necturus Kidney with 0.1 M Sodium Phosphate Buffer Solutions. pH of Glomerular Fluid (Micro) Compared with That of Perfusion Fluid

(Macro)

Animal No. ----

pH of perfusion fluid pH of glomerular fluid

1 6.75 6.80 2 7.33 7.32 3 6.94 6.90 4 6.95 6.97 5 7.17 7.17 6 6.74 6.75 7 7.02 7.04 8 6.25 6.26

A series of sixteen experiments on living Necturi was made in February and March, 1934. They included thirty-seven pH estimations. An abbreviated protocol of a typical experiment follows.

February 13, 1934. Adult male Necturus. Anesthetized, 1.45-2.00 p.m. Preparation completed 2.40 p.m. Gill movements, heart action, and glo- merular circulation good. Collection of glomerular urine and oil drip on kidney begun at 2.42 p.m. Temperature of oil 21”.

Time I E. M. F. PfI

p. m. volt 2.45 0.0086 2.47 0.0106 2.48 0.0108 2.50 0.0119 2.52 0.0114 2.54 0.0115

7.61

7.58

At 2.55 p.m., the pipette was withdrawn from the capsule, the fluid dis- charged, and the pipette rinsed twice with distilled water. At 2.58 p.m. another capsule was punctured and a second collection begun.

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770 Microquinhydrone Electrode

The results of the sixteen experiments are summarized in Table III. The pH values of glomerular urine varied from 7.07 to 7.63, with an average of 7.36.

These figures are significantly lower than those derived from the calorimetric determinations made in April, May, and June of 1933 in which the range was 7.32 to 7.67, average 7.54. The ques-

TABLE III

Determinations of pH of Glomerular Urine oj Necturus Made with Micro- quinhydrone Capillary Electrode

Animal No.

T -

Time after first deter- mination

min.

50

40 65

105

65 120 165 195

45

55

30

75 135

-

PH Animal No.

7.38 7.36 7.61 7.63 7.55 7.49 7.41 7.30 7.34 7.27 7.35 7.35 7.31 7.42 7.47 7.47 7.50 7.21 7.31 7.22

___-

8

9

10

11

12 13 14 15 16

-

- Time after first deter- mination

min.

75 100 130 200

50

75

100 350

PH

7.46 7.46 7.28 7.22 7.30 7.38 7.28 7.34 7.25 7.17 7.07 7.13 7.32 7.51 7.45 7.49 7.25

tion whether this difference represents physiological variations or is the result of undetected faults of technique seemed important. In support of the former alternative are the wide variations present in each series, the fact that the two series were made at different seasons of the year, and the occasional observation that the pH of blood and glomerular urine may undergo progressive decrease during the course of an experiment, particularly as the condition of the animal deteriorates.

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J. A. Pierce and H. Montgomery 771

It seemed necessary, however, to undertake further control experiments in which as direct comparison as possible should be made between the microelectrometric and the microcolorimetric methods. They are as follows:

The phosphate buffer solutions previously used in testing both micromet,hods were 0.066 M and 0.05 M. It was conceivable that the concentration was so great as to have masked the effect of minute traces of acid which, if present in the quinhydrone, would affect measurements of pH of the much less strongly buffered glomerular urine of Necturus. Accordingly a series of tests wit,h 0.0025 M phosphate buffer solution was made.’ Phenol red was added to make 10 mg. per cent. The buffer value of this is negli-

TABLE IV 0.00!.?5 Y Phosphate Bufer Solutions. pH Determinations by Macro- and

Microelectrometric and Microcolorimetric Methods

Electrometric Calorimetric

Macro Micro

7.13 7.16 7.16 7.24 7.23 7.25 7.29 7.30 7.32 7.12 7.14 7.10 7.18 7.20 7.21 7.17 7.15 7.14

gible in comparison with that of the phosphate. Calorimetric determinations of pH were made by the capillary tube method described in the preceding paper, and by both the macro- and microquinhydrone electrode methods as used in the experiments already described. The figures (Table IV) show no significant differences in the results of the three methods.

A protein-free ultrafiltrate of Necturus plasma, containing 10

7 It is assumed that the main buffer in glomerular urine is bicarbonate, and that bicarbonate is present in approximately the same concentration as in the plasma. The total CO2 of Necturus plasma was determined by the method of Van Slyke and found to average 20 volumes per cent. The buffer value of this concentration of COz at pH 7.4 is 9 mM per liter; that of a 0.0025 M phosphate solution at pH 7.4 is 4 mM per liter (calculated accord- ing to Van Slyke (7)).

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772 Microquinhydrone Electrode

mg. per cent of phenol red, was equilibrated with 1.5 per cent CO2 and delivered into a small glass container which was then closed with a rubber dam, air being excluded. One end of the container had previously been drawn out into a slender tube and filled with saturated KCl-2 per cent agar jelly. The liquid junction was made by dipping this tube into the saturated KC1 solution into which the side arm of the calomel half-cell projected. The micro- quinhydrone pipette was thrust through the rubber dam and the pH measurement made. At the same time samples of t,he fluid were taken into quartz capillary collecting pipettes for micro- calorimetric estimation of pH. The results of the two methods agreed within 0.02 (average) pH unit (Table V).

pH measurements with the microquinhydrone electrode show an acid drift after the fluid has been in contact with the electrode

TABLE V

UltrajLltrate from Necturus Plasma. QH Determination by Two Micromethods

Sample No. Electrometric Calorimetric

1 7.37 7.32 2 7.39 7.40 3 7.37 7.35 4 7.47 7.46 5 7.25 7.25

for a time. Experiments were made to decide whether this could have been a factor in the discrepancy between the calorimetric and electrometric glomerular urine determinations. The micro- quinhydrone electrode was inserted into a renal corpuscle of Necturus, and fluid taken in amount sufficient to cover the quin- hydrone. E.M.F. readings were made at frequent intervals for an hour or more. In every case the reading remained constant for 15 minutes after contact between fluid and electrode had been made. The drift began in from 15 to 45 minutes. Study of the time records of the earlier electrometric determinations of glomerular urine pH gave little reason to believe that such a drift had in- fluenced them.

Finally a pipette was constructed with which both calorimetric

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J. A. Pierce and H. Montgomery 773

and electrometric measurements could be made on the same sample of fluid. It consisted of a quartz pipette of 0.3 to 0.4 mm. inside diameter with a marked narrowing of the lumen 5 mm. from the pointed tip. The quinhydrone eleetrode wire was inserted to this

TABLE VI

0 0065 M Buffer Solutions. pH o.f Same Sample Determined by Two Micro-methods - ___.

Ssmple No.

1 2 3 4 5 6 7 8 9

10 11

-

--

-

Electrometrio Calorimetric --

7.14 7.18 7.21 7.17 7.23 7.20 7.20 7.15 7.21 7.24 7.27 7.22 7.23 7.21 7.23 7.21 7.28 7.26 7.29 7.26 7.22 7.19

-

TABLE VII

Glomerular Urine from Necturus. pH of Same Sample Determined by Two Microm,ethods

Animal No. -

1

2 3 4

5

Electrometric I Colorimetrie

7.39 7.43 7.60 7.57 7.61 7.6 7.37 7.42 7.41 7.43 7.37 7.41 7.61 7.59 7.55 7.58 7.56 7.59

constriction and the entire pipette filled with mercury. 0.0025 M

phosphate buffer solutions containing phenol red were drawn into such pipettes. As soon as the fluid had passed the constriction and had made effective contact with the electrode, the E.M.F.

Iaeading was made. The pipette was then removed and comparison

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774 Microquinhydrone Electrode

made between the color of the fluid in the part between the con- striction and the point and those of 0.05 M phosphate buffer stand- ards, containing the same concentration of phenol red, in capillary tubes of the same diameter. The pH values by the two methods agreed (Table VI).

Similar electrode pipettes were used in collecting glomerular urine from living Necturi which had previously been injected with phenol red subcutaneously. The time required to fill the pipette to the level of the quinhydrone was about 10 minutes. The two determinations of pH were made as in the tests with buffer solu- tions. The results by the two methods applied to the same sample of glomerular urine agreed within 0.03 (average) pH unit (Table VII).

In each of the comparisons of the electrometric with the colori- metric method described above the person who made the determi- nation by one method did not learn of the result obtained by the other until both results had been recorded.

From these results we conclude that the microcolorimetric and the microelectrometric method, when applied to the same sample of artificial solution or of glomerular urine, give essentially the same result. Hence the finding that the average pH of glomerular urine determined in one series of animals by one method- differed from that obtained in another series by the other method is to be ascribed chiefly to differences in the physiological conditions of the animal studied.

SUMMARY

A microquinhydrone electrode has been constructed with which reliable determinations of pH can be made with 0.1 c. mm. of fluid or less. It can be inserted through or into tissues without contam- ination, and escape of CO2 from the fluid which is brought into contact with it is prevented. Its accuracy is of the same order as that of the ordinary quinhydrone electrode.

The microelectrode has been used in measuring the pH of glomerular urine from Necturi. In two series of experiments forty-six determinations were made on material collected from twenty-one animals; the values ranged from 7.07 to 7.63, average, 7.39. The difference between this average value and that previ- ously obtained by a microcolorimetric method is not due to tech-

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J. A. Pierce and H. Montgomery 775

nical faults but represents physiological differences; for when both methods were applied to the same sample of glomerular urine the results agreed closely.

Grateful acknowledgment is made to Dr. William C. St>adie and to Dr. W. Mansfield Clark for much helpful advice.

BIBLIOGRAPHY

1. Wearn, J. T., and Richards, A. N., Am. J. Physiol., 71, 209 (1924). 2. Cullen, G. E., and Biilmann, E., J. Biol. Chem., 64,727 (1925). 3. Cullen, G. E., J. Biol. Chem., 63, 535 (1929). 4. Montgomery, H., J. Biol. Chem., 110, 749 (1935). 5. Clark, W. M., The determination of hydrogen ions, Baltimore, 3rd edi-

tion, 314,672, 674 (1928). 6. Hastings, A. B., and Sendroy, J., Jr., J. Biol. Chem., 61, 695 (1924). 7. Van Slyke, D. D., J. Biol. Chem., 62,525 (1922).

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J. A. Pierce and Hugh MontgomeryGLOMERULAR URINE OF NECTURUSTHE DETERMINATION OF THE pH OFELECTRODE: ITS APPLICATION TO

A MICROQUINHYDRONE

1935, 110:763-775.J. Biol. Chem. 

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