THE COLORIMETRIC ESTIMATION OF THE HYDROGEN ION CONCENTRATION OF URINE.*

BY VICTOR C. MYERS AND EDWARD MUNTWYLER.

(From the Department of Biochemistry, State University of Iowa, Iowa City.)

(Received for publication, March 21, 1928.)

Until very recently the calorimetric method of Henderson and Palmer (1) has been quite universally employed in estimating the pH of urine. Since the introduction of the sulfonephthalein series of dyes by Clark and Lubs (2) in 1917, these indicators have gradu- ally replaced the indicators employed by Henderson and Palmer. The method was modified by Palmer, Salvesen, and Jackson (3) by making the urine dilution on a smaller scale and introducing phenol red for the pH range between 6.3 and 7.4 and methyl red for the pH range between 4.7 and 6.3. Fiske (4) employed the indicators methyl red, brom-cresol purple, phenol red, and cresol red. Myers and Booher (5) suggested a simple technique of esti- mating the pH of urine in which application was made of the bi- calorimeter (6) and the phthalein dyes, phenol red, brom-cresol purple, and brom-cresol green. Wedges were calibrated for each of these indicators, the range employed for phenol red being pH 6.6 to 8.4, for brom-cresol purple pH 5.2 to 7.0, and for brom- cresol green pH 4.6 to 5.4. The last named sulfonephthalein dye had recently been introduced by Cohen (7) to replace methyl red. Estimations were made at room temperature on urine diluted 1:lO with distilled water. It was then pointed out that so far as the matching of colors with the bicolorimeter went the error should not exceed f pH 0.02 to 0.04, but it was realized that the factors of temperature, dilution, salt, and COZ content of urine

* The data here presented are taken from a Thesis in Chemistry by Ed- ward Muntwyler submitted to the Graduate College of the State University of Iowa, July, 1927, in partial fulfilment of the requirements for the degree of Master of Science.

225

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226 Calorimetric Estimation of pH of Urine

might introduce a very appreciable error in the determination. Accordingly a study of these factors was undertaken in which the electrometric method was employed as a check. A preliminary report of these observations was made by Muntwyler, iSorris, and Myers (8). While this work was in progress Hastings, Sen- droy, and Robson (9) made a report covering many of these points. Their calorimetric estimations were made at 38” on urine diluted 1: 5. They have employed bicolor standards consisting of a series of paired tubes containing the acid and alkaline forms of the indicator in proportions corresponding to the pH. In spite of the fact that they carefully controlled the factors which might introduce error, they noted considerable divergence from the elec- trometric values. By subtracting 0.1 pH from the observed calorimetric value their figures agreed within f 0.1 pH of the true value. They conclude that at a fivefold dilution the correction involved in the determination may be attributed almost entirely to the effect of dilution.

It is ordinarily much less convenient to make color compari- sons at 38” than at room temperature, and, although some of our studies have been carried out at 38”, we have directed our efforts particularly to obtaining a correction factor for calorimetric de- terminations made at room temperature on diluted urine, com- parison being made with values obtained electrometrically at 38” on the undiluted sample. The method of Myers and Booher (5) has been modified in several particulars in an attempt to elimi- nate as far as possible such errors as the effect of dilution and loss of coz. As pointed out by Marshall (lo), the loss of CO2 from urine may introduce a considerable error in the determination of pH, particularly where the latter is high. A saline indicator mixture is employed as a diluent for each indicator and set at a definite pH. Although satisfactory calorimetric readings may be made with the bicolorimeter on a large number of urine samples without dilution, this is not always true. On this account the urine is uniformly diluted 1:5. We have adopted the 1:5 dilu- tion of Hastings, Sendroy, and Robson (9) but believe that the use of a saline solution in place of water increases the accuracy of the determination. Since urine samples may vary widely in their salt content, much consideration has been given to the effect of salt on the indicators. Use has been made of the following indi-

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V. C. Myers and E. Muntwyler 227

caters: phenol red, brom-thymol blue, brom-cresol purple, and brom-cresol green. Where the highest degree of accuracy is not needed it is feasible to omit the use of brom-thymol blue, since with the bicolorimeter it is possible to match the border line colors satisfactorily in the pH range where phenol red and brom- cresol purple meet.

Method.

Collection of Urine.-The urine is collected under oil in Pyrex Erlenmeyer flasks with the aid of long stemmed funnels.

Diluting Fluids.-As a diluent a saline indicator mixture is prepared as follows: To 780 cc. of 0.9 per cent sodium chloride solution, 100 cc. of the desired diluted indicator are added. This, when diluted, gives the same concentration of dye as in the un- known. Brom-cresol purple, brom-thymol blue, and brom-cre- sol green in 0.04 per cent strength are prepared from 0.4 per cent stock solutions’ and phenol red in 0.02 per cent from 0.4 per cent stock solution made up as recommended by Clark (11). These mixtures are then adjusted to a definite pH with the aid of minute quantities of strong solutions of NaOH or HCl. For phenol red the mixture is adjusted to pH 7.4, while the brom-thymol blue mixture is set at pH 7.2, the brom-cresol purple at pH 6.2, and the brom-cresol green at pH 5.0. These solutions are preserved in Pyrex saline bottles as recommended by Myers, Schmitz, and Booher (12).

Preparation of Standard Wedges.-Two wedges are prepared for each indicator, one containing an alkaline and the other an acid solution of the indicator. The alkaline wedges employed for phenol red, brom-cresol purple, and brom-thymol blue are filled with ~/15 secondary phosphate containing 2 cc. of the corresponding diluted indicator for 20 cc. of phosphate. The acid wedges for these indicators are filled with ~/15 primary phosphate to which a similar amount of indicator has been added. The wedges for the brom-cresol green indicator are made up with Clark’s phthal- ate-NaOH mixtures, the alkaline wedge having a pH of about

1 These are prepared by grinding up the required amount of dye with the molecular amount of NaOH, the amount of alkali needed for each decigram of dye being: 5.7 cc. of 0.05 N NaOH for phenol red, 3.2 cc. for brom-thymol blue, 3.7 cc. for brom-cresol purple, and 3.2 cc. for brom-cresol green.

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228 Calorimetric Estimation of pH of Urine

6.2 and the acid wedge of about 4.0. As above, 2 cc. of the diluted indicator are used for 20 cc. of the buffer mixture. 10 (or 5) cc.

TABLE I.

Buffer Mixtures for Calibration of Wedges.

PH M/10 M/10

CH phthalatc. NaOH.

4.0 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 6.2

cc. cc.

5 0.04* 5 1.22 5 1.50 5 1.77 5 2.08 5 2.39 5 2.68 5 3.00 5 3.26 5 3.55 5 4.70

&x/15 KHsPOd. a/15 NmHPO4

5.2 9.82 0.18 5.3 9.74 0.26 5.4 9.64 0.36 5.5 9.58 0.42 5.6 9.48 0.52 5.7 9.33 0.67 5.8 9.16 0.84 5.9 9.00 1.00 6.0 8.71 1.29 6.1 8.40 1.60

PH

6.2 6.3 6.4 6.5 6.6 6.7 6.3 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0 8.1 8.2 8.3 8.4

M/15 XHzPOc.

cc. cc.

8.09 1.91 7.74 2.26 7.30 2.70 6.82 3.18 6.30 3.70 5.66 4.34 5.08 4.92 4.48 5.52 3.89 6.11 3.34 6.66 2.80 7.20 2.32 7.63 1.92 8.08 1.59 8.41 1.30 8.70 1.06 8.94 0.85 9.15 0.69 9.31 0.56 9.44 0.43 9.57 0.32 9.68 0.25 9.75 0.20 9.80

* The phthalate-NaOH mixtures are made up to a volume of 10 cc. The Serensen buffer phosphate standards from pH 5.2 to 7.8, prepared

as in the table, have been found to check electrometrically within the limits of experimental error. As will be seen from Table II the molarity of these standards is important.

The 1x/15 phosphate solutions are prepared from special reagent salts (Merck’s are satisfactory). The secondary sodium phosphate is prepared by dissolving 9.47 gm. of anhydrous Na2HP04 in distilled water and making up to 1 liter, while the primary potassium phosphate is similarly prepared from 9.08 gm. of KHnPOa.

portions of the different phosphate and phthalate-NaOH mixtures are prepared as given in Table I and 1.0 (or 0.5) cc. of the corre-

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V. C. Myers and E. Muntwyler 229

sponding indicator added. Readings are then obtained for each set of wedges with the dominant color nearer the eyepiece. By plotting the scale reading against the pH value a calibration curve for each set of wedges is obtained. Phenol red is calibrated be- tween pH 7.0 and 8.0, brom-thymol blue between 6.4 and 7.4, brom-cresol purple between 5.4 and 6.6, and brom-cresol green between 4.6 and 5.4. Standards are made up differing by 0.1 pH. One should check the wedges against new standards every 2 weeks. When the wedges have been carefully calibrated it is probably sufficient to refill them once in 2 weeks with the same freshly prepared buffer solutions.

Calorimetric Readings and Calculation of Results.-Since the majority of urines have a pH occurring in the brom-cresol purple range, this indicator is tried first. 1.6 cc. of the brom-cresol purple indicator-saline mixture are allowed to flow into the cup of the bicolorimeter under oil. Enough urine is drawn under oil into a 1 cc. syringe so that 0.4 cc. may be transferred into the saline in the cup. The solution is then thoroughly stirred with the needle of the syringe and color comparison made. If the color of the brom-cresol purple indicates an acid urine the brom-cresol green system is employed. On the other hand if the color of the brom- cresol purple indicates an alkaline urine either the brom-thymol blue or the phenol red systems may be employed depending upon the degree of alkalinity. By subtracting 0.2 pH from the observed calorimetric value at 25” the true pH of the undiluted urine at 38” as determined electrometrically is obtained to within & 0.1 pH. Urine becomes more acid as the temperature is raised by approxi- mately 0.01 pH per 1” and hence slight differences in room tem- perature may be corrected to 25”.

EXPERIMENTAL.

Since it was felt that with the calorimetric method as outlined the effect of loss of COz was at a minimum and the effect of dilution was stabilized as far as possible, any abnormal deviations between the calorimetric and electrometric values could be explained by the effect of salt on the indicator. The effect of varying salt concen- trations on the indicator was thus considered in some detail.

In every case the electrometric estimations were made on the

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230 Calorimetric Estimation of pH of Urine

undiluted specimen. A modified Clark electrode vessel (Pyrex glass) of 2 cc. capacity with a thermometer opening directly into the vessel was employed. Determinations at 38” were made in an air thermostat. The e of the calomel cell was calculated from the voltage obtained from 0.1 N HCl to which was assigned a pH value of 1.085 at 38”. All potential measurements were corrected

TABLE II.

Comparison of Electrometric and Calorimetric pH Values in Solutions of Varying Phosphate Concentration.

Phosphate. pH,25’= - pHc25’.’ Indicator.

M

0.20 0.10 0.067 0.05 0.033 0.02 0.01 0.005 0.063 0.10 0.067 0.05 0.03 0.02 0.01

0.005

-0.10

-0.04 0.00 0.02 0.05 0.09 0.13 0.17 0.20

-0.02 0.00 0.055 0.075 0.12 0.15 0.19

Phenol red. ‘I “ “ “ ‘I “ “ “ I‘ “ “ “ “ “ “ CL

Brom-cresol purple. ‘I “ “ “ “ ‘I “ ‘I “ “ “ “

* These figures are an average of a considerable number of determina- tions covering the whole useful range of phenol red. In the case of brom- cresol purple the determinations were made close to pH 6.0. The divergence is due in large part to the change in electrometric values, the calorimetric pH remaining relatively constant.

to 760 mm. of dry hydrogen. For the comparative study, urine samples were equilibrated with 40 mm. of CO, and then placed under oil. Samples of these were taken for the calorimetric and electrometric estimations. It was found that reproducible volt- ages could be obtained with one or two refills if a CO2 tension of 40 mm. was introduced into the electrode vessel with the hydrogen.

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V. C. Myers and E. Muntwyler 231

TABLE III.

Comparison of Calorimetric and Electrometric pH Values. Sodium Chloride Varied in a Given Phosphate Concentration.

Phosphate. N&l pK, 25o.a pH, 25’. PH, - PH,

Y M

0.085 0.050 0.085 0.100 0.085 0.125 0.085 0.150 0.085 0.200 0.085 0.250 0.085 0.375 0.085 0.500

7.46 7.44 7.55 7.42 7.40 7.48 7.44 7.41

0.043 0.050 7.43 0.043 0.100 7.42 0.043 0.125 7.53 0.043 0.150 7.39 0.043 0.200 7.37 0.043 0.250 7.47 0.043 0.375 7.45 0.043 0.500 7.41

7.41 0.05 7.37 0.07 7.46 0.09 7.34 0.08 7.30 0.10 7.36 0.12 7.32 0.12 7.25 0.15

7.44 -0.01 7.40 0.02 7.49 0.04 7.34 0.05 7.29 0.08 7.38 0.09 7.31 0.14 7.26 0.15

0.020 0.050 7.50 7.51 -0.01 0.020 0.100 7.45 7.45 0.00 0.020 0.125 7.50 7.44 0.06 0.020 0.150 7.43 7.39 0.04 0.020 0.200 7.42 7.34 0.08 0.020 0.250 7.44 7.28 0.16 0.020 0.500 7.39 7.20 0.19

0.008 0.050 7.54 7.50 0.04 0.008 0.125 7.45 7.39 0.06 0.008 0.250 7.39 7.28 0.11 0.008 0.500 7.31 7.13 0.18

0.005 0.050 7.42 7.41 0.01 0.005 0.125 7.38 7.33 0.05 0.005 0.250 7.28 7.18 0.10 0.005 0.500 7.25 7.08 0.17

- * In the calorimetric estimation 1 cc. of the dilute indicator solution was

added to 10 cc. of the phosphate-sodium chloride solutions.

- Phenol red.

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232 Calorimetric Estimation of pH of Urine

Phosphate. 1

TABLE III-~onduded.

NaCl pH, 25’.* pH, 25’. PH, - ~6.

Brom-cresol purple.

dl

0.057 0.057 0.057

0.033 0.033 0.033 0.033 0.033 0.033

0.020 0.020 0.020 0.020

0.01 0.01 0.01

0.01

0.008

0.008

0.008

0.008

M

0.110

0.220 0.330

6.17 6.11 0.06 6.11 5.99 0.11

6.02 5.87 0.16

0.050 6.06 6.06 0.00 0.110 6.17 6.11 0.06 0.125 6.02 5.95 0.07 0.220 6.11 5.99 0.12 0.330 6.05 5.87 0.18 0.350 5.91 5.77 0.14

0.050 6.10 6.10 0.125 6.04 5.99 0.250 6.00 5.87 0.500 5.92 5.71

0.050 6.11 6.13 0.125 6.04 6.00 0.250 5.99 5.87 0.500 5.93 5.71

0.050 6.13 6.14 0.125 6.09 6.00 0.250 6.01 5.87 0.500 5.92 5.71

0.00 0.05 0.13 0.21

-0.02 0.04 0.12 0.22

-0.01

0.09

0.14

0.21

It has ordinarily been assumed that dilution has little effect on a well buffered solution. The directions given for calorimetric determinations of urinary pH call for dilutions from 1:5 to 1:25. In this connection the data of Table II are interesting. At 0.06 M, the same strength as used in the wedges, the calorimetric and electrometric values correspond. However, as the molar strength becomes less than 0.067 the electrometric assumes the greater value, while as the molar strength becomes greater the colori- metric assumes the greater value.

In order to see what magnitude of effect the salt would have on the indicator color, various solutions were prepared having vari- able sodium chloride and uniform phosphate concentrations.

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V. C. Myers and E. Muntwyler 233

Calorimetric and electrometric values were obtained on these solutions at 25”. The calorimetric values were obtained by mak- ing the color comparisons on solutions to which 1 cc. of indicator had been added to 10 cc. of solution. Table III shows the com- parison for phenol red and brom-cresol purple. It will be noted that as the concentration of salt increases, the deviation between the calorimetric and electrometric values increases. Lepper and Martin (13) have already made this same observation for phenol red.

To make a system with which the salt effect of the indicator could be studied and have the results comparable to what might be found in urine, solutions of urea, sodium chloride, phosphate, and sodium bicarbonate were prepared. By keeping the concen- tration of three of the constituents constant the effect of the fourth could be studied. The solutions were equilibrated with 40 mm. of COz and then placed under oil. Electrometric determinations were made at 38” on the undiluted solution. Calorimetric values were obtained both on the diluted and undiluted sample at 25”. The calorimetric values for the diluted samples were obtained by the method employed for urine; i.e., 0.4 cc. added to 1.6 cc. of saline under oil. The other calorimetric value was obtained by adding 1 cc. of indicator to 10 cc. of solution under oil. Table IV gives the result of this study on brom-cresol purple, brom-thymol blue, and phenol red indicators. As the concentration of salt increases, the deviation between the electrometric and calorimetric values also increases. The deviation for the diluted samples is greater than that of the undiluted at the higher salt concentrations. In the concentrations studied urea does not appear to influence the colori- metric value. Since the sodium chloride concentration may vary markedly in urine, one might expect variations in the differences between the electrometric and calorimetric pH values of similar magnitude as with the prepared solution. If such were the case no absolute correction factor could be applied to the urine.

Since emphasis has been placed on the concentration of sodium chloride in the prepared solutions, it has been determined in urine samples along with the calorimetric and electrometric pH. Table V gives the results of this study. As may be seen, in spite of the fact that the salt concentration varied quite widely, there is no clear cut relation between it and the difference between the calorimetric

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TABLE IV.

Comparison of Electrometric and Calorimetric pH Values in Varying C’oncen- trations of Urea, Sodium Chloride, Sodium Bicarbonate, and Phosphate.

PO4 N&l UP%% NaHCOa

pH, 25’ pH, 25” (undiluted) (dilutedl:5*) - pH, 38O - pH, 38”

(undiluted). (undiluted).

Phenol red.

M

0.01 0.01 0.01 0.01 0.01

0.01 0.01 0.01 0.01

0.01 0.01 0.01 0.01

0.01 0.01 0.01 0.01

0.01 0.01

0.01 0.01 0.01 0.01 0.01

0.02 0.02 0.02 0.02 0.02

-

-

- *Diluted 1

M

0.025 0.05 0.10 0.20 0.30

0.075 0.150 0.225 0.30

0.125 0.125 0.125 0.125

0.10 0.10 0.10 0.10

0.20 0.20

0.0625 0.10 0.20 0.25 0.375

0.0625 0.10 0.20 0.25 0.375

-

-

-

dl

0.375 0.375 0.375 0.375 0.375

0.375 0.375 0.375 0.375

0.375 0.375 0.375 0.375

0.625 0.500 0.625 0.250

0.500 0.125

db

0.01 0.01 0.01 0.01 0.01

0.015 0.015 0.015 0.015

0.005 0.015 0.02 0.025

0.01 0.01 0.01 0.01

0.01 0.01

- I

Brom-thymol blue.

0.3 0.3 0.3 0.3 0.3

0.3 0.3 0.3 0.3 0.3

0.007 0.007 0.007 0.007 0.007

0.007 0.007 0.007 0.007 0.007

5 with 0.9 per cent NaCl solution.

234

-0.01 -0.01 0.05 0.07 0.07 0.11 0.15 0.20 0.21 0.27

0.16 0.15 0.14 0.17 0.18 0.22 0.22 0.27

0.15 0.13 0.11 0.16

0.17 0.13 0.13 0.15

0.12 0.13 0.12 0.14 0.13 0.16 0.13 0.16

0.16 0.20 0.17 0.22

-0.03 -0.04 0.06 0.06 0.10 0.11 0.12 0.18 0.16 0.26

0.06 0.04 0.09 0.12 0.14 0.18 0.16 0.24 0.19 0.30

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TABLE IV-CO?&bdt?d.

PO4

pH, 25’ pH, 25”

N&l Ur.Z+. NaHCOs (undiluted) (diluted 1:5*) - pH, 38” - pH, 38”

(undiluted). (undiluted).

Brom-thymol blue-Continued.

M

0.02 0.02 0.02 0.02

M M

0.125 0.00

0:125 0.30 0.125 0.50 0.125 0.60

0.06 0.09 0.07 0.08 0.05 0.08 0.04 0.07

0.01 0.01 0.01 0.01 0.01

0.02 0.02 0.02 0.02 0.02

0.03 0.03 0.03 0.03 0.03

0.02 0.02 0.02 0.02 0.02

0.02 0.02 0.02 0.02 0.02

0.02 0.02 0.02 0.02 0.02

0.05 0.125 0.0025 0.01 -0.04 0.10 0.125 0.0025 0.05 0.04 0.20 0.125 0.0025 0.12 0.14 0.25 0.125 0.0025 0.14 0.17 0.375 0.125 0.0025 0.19 0.25

0.05 0.125 0.0025 0.01 0.01 0.10 0.125 0.0025 0.06 0.07 0.20 0.125 0.0025 0.11 0.15 0.25 0.125 0.0025 0.14 0.19 0.375 0.125 0.0025 0.18 0.26

0.05 0.125 0.0025 0.03 0.02 0.10 0.125 0.0025 0.06 0.06 0.20 0.125 0.0025 0.08 0.13 0.25 0.125 0.0025 0.12 0.18 0.375 0.125 0.0025 0.15 0.24

0.10 0.10 0.10 0.10 0.10

0.0025 0.08 0.08 0.0025 0.08 0.06 0.0025 0.06 0.06 0.0025 0.07 0.06 0.0025 0.08 0.06

0.20 0.20 0.20 0.20 0.20

0.0025 0.12 0.17 0.0025 0.12 0.16 0.0025 0.11 0.16 0.0025 0.11 0.15 0.0025 0.13 0.18

0.25 0.25 0.25 0.25 0.25

0.05 0.10 0.20 0.30 0.375

0.05 0.10 0.20 0.30 0.375

0.05 0.10 0.20 0.30 0.375

0.0025 0.15 0.20 0.0025 0.15 0.21 0.0025 0.14 0.23 0.0025 0.16 0.21 0.0025 0.14 0.20

Brom-crest ,urple. - -

- 235

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236 Calorimetric Estimation of pH of Urine

TABLE V.

Comparison of Calorimetric and Electrometric pH Values of Urine Specimens Arranged According to the Molarity of the Sodium Chloride.

1

Specimen No. NaCl pH, 25” pH, 33”

(diluted 1:5*). (undihted). PH, - PH,.

Phenol red.

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15

M

0.020 0.061 0.065 0.089 0.093 0.100 0.110 0.154 0.190 0.200 0.212 0.222 0.226 0.246 0.300

7.02 6.78 0.24 7.15 6.89 0.26 7.09 6.88 0.21 7.22 7.05 0.17 7.23 7.00 0,23 7.10 6.87 0.23 7.38 7.10 0.28 7.34 7.06 0.28 7.26 7.06 0.20 7.55 7.33 0.22 7.66 7.39 0.27 7.28 7.22 0.26 7.11 6.83 0.28 7.40 7.06 0.34 7.50 7.22 0.28

Brom-thymol blue. - 1 0.034 6.55 6.49 0.09 2 0.065 6.56 6.42 0.14 3 0.0139 7.22 7.05 0.17 4 0.090 6.99 6.70 0.19 5 0.100 6.72 6.53 0.19 6 0.103 6.97 6.73 0.24 7 0.110 6.98 6.75 0.23 8 0.154 6.83 6.75 0.08 9 0.157 6.74 6.53 0.21

10 0.190 7.30 7.06 0.24 11 0.230 6.98 6.75 0.23 12 0.230 6.88 6.68 0.20

- Brom-cresol purple.

0.051 6.00 5.80 0.20 0.058 6.13 5.96 0.17 0.065 6.23 6.10 0.13 0.085 6.56 6.33 0.23 0.093 5.90 5.73 0.17

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TABLE V-Continued.

Specimen No. N&l pH, 25’ pH, 38”

(diluted 1:5*). (undiluted). PHC - PH,

Brom-cresol purple-Continued.

6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

M

0.099 0.102 0.110 0.116 0.120 0.120 0.130 0.140 0.144 0.147 0.150 0.160 0.164 0.168 0.180 0.191 0.195 0.200 0.201 0.216 0.230 0.230 0.236 0.230 0.246 0.250 0.274 0.277

6.14 5.99 0.15 6.40 6.25 0.15 6.32 6.28 0.04 5.77 5.52 0.25 6.01 5.82 0.19 6.42 6.29 0.13 6.03 5.82 0.21 5.74 5.54 0.20 6.28 6.03 0.25 6.62 6.44 0.18 6.42 6.23 0.19 6.20 5.98 0.22 6.68 6.53 0.15 5.83 5.58 0.25 6.14 5.96 0.18 6.30 6.09 0.21 6.35 6.15 0.20 6.23 5.89 0.34 5.69 5.42 0.27 6.16 5.96 0.20 6.21 5.98 0.23 5.85 5.62 0.23 6.07 5.82 0.25 6.05 5.82 0.23 6.45 6.18 0.27 5.87 5.63 0.24 6.12 5.82 0.30 5.91 5.73 0.18

Brom-cresol green.

1 0.157 5.12t 2 0.168 5.09 3 0.177 5.09 4 0.185 5.21 5 0.194 5.16 6 0.198 5.18 7 0.210 5.20

-

-

4.91 0.21 4.84 0.25 4.90 0.19 4.97 0.24 4.95 0.21 4.96 0.22 4.98 0.22

* Diluted 1 : 5 with 0.9 per cent sodium chloride solution containing the respective indicators.

t Calorimetric values for brom-cresol green above 25” corrected to 25” by adding 0.01 pH per 1”.

237

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238 Calorimetric Estimation of pH of Urine

TABLE V-Concluded.

Summary.

Average correction for phenol red -0.24 pH. 12 out of 15 are within &to. 05 pH of the average.

Average correction for brom-thymol blue -0.20 pH. 9 out of 12 are within f0.05 pH of this value.

Average correction for brom-cresol purple -0.205 pH. 26 out of 33 are within ~0.05 pH of the average. Average correction for Specimens 1 to 17, 0.17 pH; Specimens 18 to 33,0.23 pH.

Average correction for brom-cresol green -0.22 pH. Maximum devia- tion in 7 determinations ~0.03 pH of the average correction.

TABLE VI. Comparison of Calorimetric and Electrometric pH Values of Urine.

1 2 3 4 5 6 7 8 9

10 11 12

-

- _

-

N&l pH, 38”. pHe 38”. ‘Hc - PI& Indicator.

M

0.048 0.072 0.085 0.089 0.109 0.120 0.160 0.190 0.208 0.284 0.058 0.085

5.99 5.95 0.04 6.15 6.08 0.07 6.41 6.28 0.13 6.13 6.06 0.07 6.12 6.01 0.11 6.27 6.13 0.14 6.35 6.23 0.12 6.37 6.16 0.21 5.95 5.79 0.16 6.10 5.92 0.18 7.14 7.06 0.08 7.30 7.24 0.06

Brom-cresol purple. ‘I “ ‘I “ “ “ “ ‘I I‘ ‘I “ “ “ ‘I “ “ “ “

Phenol red. “ “

Average difference.................. 0.11

and electrometric values. However, in the brom-cresol purple series it will be noted that the first seventeen urine specimens with the lowest sodium chloride concentration showed an average difference of 0.17 pH, while the last sixteen specimens with higher salt concentrations showed an average difference of 0.23pH. The average correction for the brom-cresol purple and brom-thymol blue is 0.20 pH, while that for the phenol red and brom-cresol green is slightly higher; namely, 0.24 and 0.22 pH respectively.

A few determinations, shown in Table VI, were made at 38”

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V. C. Myers and E. Muntwyler 239

both electrometrically and calorimetrically. The average differ- ence between the two values is 0.11 pII. Using the bicolorimeter at 38” was found to be less convenient than at room temperature, since the instrument must be placed in the thermostat with the eyepiece protruding. It is hence recommended that the deter- minations be made at room temperature (as close to 25” as pos- sible), slight changes in temperature from 25” being corrected with the aid of Table VII.

TABLE VII.

Changes in Urine pH with Temperature.

Specimen No. pH, 38”. pH, 25”. pH, 25’ - pH, 38”.

1 6.18 6.27 2 5.63 5.73 3 6.09 6.15 4 6.10 6.17 5 5.73 5.81 6 5.62 5.70

Average difference.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

0.09 0.10

0.06 0.07 0.08 0.08

0.05

DISCUSSION.

While it is true that an error of 0.1 or 0.2 pH in the estimation of the pH of urine is of relatively little significance owing to the wide variations which may be encountered normally (4.8 to 8.0 pH), still in carrying out any determination the error of the method should be known. When this study was undertaken we did not feel that this was true of the estimation of the urinary pH by the calorimetric method. Furthermore, we felt that a study of the calorimetric method as applied to urine might throw some light on the method as used for blood, and other body fluids, also that some of the observations might be applied to bacteriological culture media.

Attention should be called to the fact that the calorimetric value of various solutions may at times deviate quite appreciably from the electrometric value, depending upon the salt concentra- tion (Tables II, III, and IV). For example, when calorimetric and electrometric values are obtained on various strength phos- phate solutions, the deviation becomes increasingly great as the

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240 Calorimetric Estima’tion of pH of Urine

molar strength of the phosphate varies from 0.067 M. When sodium chloride is added to the phosphate solution and the molar- ity of the latter kept constant, while the concentration of sodium chloride is gradually increased, the divergence between the elec- trometric and calorimetric methods increases. The addition of urea appears to be without effect. The influence of the molarity of sodium chloride in the urine series (Table V) is not so readily apparent, but nevertheless if the first seventeen determinations in the brom-cresol purple group are compared with the last sixteen determinations, it will be observed that the divergence between the two determinations is 0.17 pH for the urines with the lowest content of sodium chloride and 0.23 pH for those with the highest content. From these observations the use of a saline diluent in place of water would seem obvious.

In studying the calorimetric change of pH on dilution of both urine and prepared solutions it was found that the value obtained depended in some degree upon the pH of the solution in respect to the pH of the diluent. A urine with a high pH diluted with distilled water having a pH of about 6.0 has a different pH dilution curve than that of a urine with a low pH. A 1: 5 dilution gave the least deviation and yet seemed to reduce color interference to a minimum. To eliminate as far as possible the variations in pH due to the variation in the pH of the diluent and to stabilize the effect of change in salt concentration on dilution, various saline indicator solutions set at a definite pH have been used. This principle was used by Cullen (14) in his calorimetric method for the estimation of the pH of blood plasma. Dilutions of 10 to 25 times with water as has been done in the past may lead in some instances to considerable error. Hastings, Sendroy, and Robson (9) em- ploy a 1:5 dilution with water and make the color comparison at 38”. This gave them an average correction of 0.1 pH. In the limited number of samples determined at 38” in this work with the saline diluent an average correction of the same magnitude was obtained, namely 0.11 pH, thus confirming their average figure.

While it may seem troublesome to prepare saline solutions for each indicator it saves time where many determinations are to be run. With the use of the special bottles the saline solutions, when once adjusted, will keep for considerable periods. After the saline solutions have been prepared, and. the wedges calibrated, check

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V. C. Myers and E. Muntwyler 241

determinations may be made within 5 minutes, and a hundred or more determinations carried out in the course of a day. Since the determinations are performed under oil and the addition of urine is rapidly accomplished, the loss of COZ is minimal. The determinations are carried out at room temperature (25”). In addition to the greater convenience of this, there would seem to be less opportunity for loss of CO2 than at 38”. When the de- terminations are carried out as described at room temperature on urine diluted 1:5 with the saline-indicator solution the results, after the correction factor is deducted, agreed in 80 per cent of the cases within 0.05 pH of the correct value of the undiluted speci- men at body temperature.

In the matching of colors a very high degree of accuracy may be secured with the bicolorimeter, quite equal to that obtained with the best models of the Duboscq calorimeter. Although modifications of the Duboscq have been suggested permitting the matching of two colors, it is believed that for this work the bi- calorimeter has inherent superiorities: The standards are kept in tightly stoppered wedges and are not exposed to the air, and one views the unknown in a horizontal instead of a vertical position thus permitting the unknown to be covered with oil without inter- fering with the action of the plunger. Since only the more alka- line wedge is calibrated the yellow of the acid wedges may be used, in part, to match out the yellow of the urine still present at the 1:5 dilution.

SUMMARY.

A calorimetric method for estimating the pH of urine is de- scribed in which use is made of the bicolorimeter and the phthal- ein dyes, phenol red, brom-thymol blue, brom-cresol purple, and brom-cresol green.

When a 1: 5 dilution with a saline diluent set at a definite pH rather than with distilled water, is employed and the determina- tion made under oil, the effect of dilution is stabilized as far as possible and the loss of CO2 is minimal.

The determinations are performed at room temperature (25”) and, by means of corrections obtained by comparison with the electrometric method, the values are corrected to 38”. The cor-

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242 Calorimetric Estimation of pH of Urine

rection factor for phenol red is 0.24, for brom-thymol blue 0.20, for brom-cresol purple 0.20, and for brom-cresol green 0.22 pH. In a study of 67 urine specimens with the pH ranging from 4.9 to 7.2 the above corrections brought the calorimetric estimations within 0.05 pH of the correct value in 80 per cent of the deter- minations.

Consideration has been given to the effect of salt on the indicator and its possible interference in the determination of the pH in urine discussed. As the salt concentration increases above ~/15 the calorimetric value is greater than the electrometric, while with concentrations less than ~/15 the reverse is true.

BIBLIOGRAPHY.

1. Henderson, L. J., and Palmer, W. W., J. Biol. Chem., 1912-13, xiii, 393. 2. Clark, W. M., and Lubs, H. A., J. Bact., 1917, ii, 1,109,191. 3. Palmer, W. W., Salvesen, H., and Jackson, H., Jr., J. Biol. Chem., 1920-

21, xiv, 101. 4. Fiske, C. H., J. Biol. Chem., 1921, xlix, 163. 5. Myers, V. C., and Booher, L. E., Proc. Sot. Exp. Biol. and Med., 1924-25,

xxii, 511. 6. Myers, V. C., J. Biol. Chem., 1922, liv, 675. 7. Cohen, B., Proc. Sot. Exp. BioZ. and Med., 1922-23, xx, 124. 8. Muntwyler, E., Norris, E. R., and Myers, V. C., Proc. Sot. Exp. BioZ.

and Med., 192526, xxiii, 826. 9. Hastings, A. B., Sendroy, J., *Jr., and Robson, W., J. BioZ. Chem., 1925,

Ixv, 381. 10. Marshall, E. K., Jr., J. BioZ. Chem., 1922, li, 3. 11. Clark, W. M., The determination of hydrogen ions, Baltimore, 2nd

edition, 1923. 12. Myers, V. C., Schmitz, H. W., and Booher, L. E., J. BioZ. Chem., 1923,

lvii, 209. 13. Lepper, E. H., andMartin, C. J., Biochem. J., 1926, xx, 45. 14. Cullen, G. E., J. BioZ. Chem., 1922, lii, 501.

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Victor C. Myers and Edward MuntwylerCONCENTRATION OF URINE

THE HYDROGEN ION THE COLORIMETRIC ESTIMATION OF

1928, 78:225-242.J. Biol. Chem. 

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