STUDIES OF ACIDOSIS.

XX. TI-IE COLORIMETRIC DETERMINATION OF BLOOD pH AT BODY TEMPERATURE WITHOUT BUFFER STANDARDS.

BY A. BAIRD HASTINGS AND JULIUS SENDROY, JR.

(From the Hospital of The Rockejeller Institute for Medi::al Research.)

(Received for publication, August 5, 1924.)

This paper has for its purpose, first, a study of the method of determining pH without buffer standards, and its application to blood pH determinations; second, a comparison of colori,metric and electrometric dctcrminations of blood pH at body tem- peraturc; and third, an analysis of the variables entering into the correction factor used by Cullen (1) to convert calorimetric pH values at 20’ into electrometric pH values at 38”.

Bjer urn (2) first introduced the bicolor principle for determining pH without standard buffer solutions, by employing two wedge-shaped glass vessels superimposed upon each other, one of which contained an indicator in acid solution, while the other contained the same indicator inalkaline solution. By looking through the superimposed wedges at different points he observed mixed colors equal to those of buffered solutions of certain deli- nite pH values. Myers (3) has recently utilized this principle in devising a wedge calorimeter which is useful for the determination of the ~11 of :mall amounts of material with a high degree of accuracy.

Gillespie (4) showed that the Clark and Lubs series of indicators could be used in this way by combining pairs of test-tubes containing the “acid form” and the “alkaline form” of the indicators in definite ratios and that the pH corresponding to each ratio was given by the mass law equation in the logarithmic form

PH = PR’ + log alkaline form

acid form

Michaelis (5) has used this principle for the determination of pH by his series of monocolor indicators, the nitrophenols and phenolphthalein.

Up to the present the bicolor principle has been employed only in pH determinations where standards at intervals of less than 0.20 pH were unnecessary. We have found that phenol red, the

G95

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696 Calorimetric Plasma pH

indicator recommended by Cullen for the calorimetric deter- mination of the reaction of blood, may be used for the preparation of standards by the bicolor method at 0.05 pH intervals, thereby permitting the estimation of calorimetric pH values to 0.02 PH. This corresponds to the accuracy possible with phosphate standards.

The advantages of the bicolor standards are stability, re- producibility, freedom from temperature effects, and simplicity of preparation.

Method.

Preparation of Standards.

The solutions required are phenolsulfonphthalein (phenol red) of known concentration, dilute alkali, e.g. 0.01 N NaOH, and dilute acid, e.g. 0.0001 N HCl. The only apparatus needed is a micro burette, a sufficient number of clear glass test-tubes of the same internal diameter, and a 3-row wooden comparator block of convenient size. The concentrations of indicator in the acid and alkaline solutions should be such that the total amount of dye in each pair of tubes is constant and provides a color depth convenient for accurate reading.

We have found the following concentrations of indicator to be satisfactory. A stock solution of phenol red, 0.1 per cent, made up as recommended by Clark (6), is kept on hand. When standard solutions are to be prepared, 15 cc. of the 0.1 per cent solution are diluted to 200 cc., yielding a 0.0075 per cent solution. In each pair of standard tubes the total amount of this indicator solution is 2.5 cc. The total volume of solution in each tube is 25 cc. Table I shows the amounts of 0.0075 per cent indicator solution to be added to the alkaline and acid tubes, respectively, to give the combined color effect corresponding to certain pH values.

The pH corresponding to each E ratio has been determined

by comparing tubes made up in the manner described above with standard phosphate solutions containing the same concentration of indicator. This comparison was made at 20” and 38°C. The results of these experiments are given in Table II and Fig. 1.

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A. B. Hastings and J. Sendroy, Jr.

The curves are drawn from the equations

697

alkaline form alkaline form p&o” = 7.78 + log acid form and P&’ = 7.65 + log

acid form

where 7.78 and 7.65 are the values of pK’ of the dye at 20” and 38”, respectively, determined in the manner outlined later. The crosses, representing the experimental readings, show complete

TABLE I.

Table of pH Values at 20” and 58” at 0.06 Intervals, with Corresponding Amounts of 0.0076 Per Cent Phenol Red and 0.01~ NaOH or 0.0001 N HCI.

pH20”

-

_ - Alkali tube. I Acid tube. ,H~EP Alkali tube. Acid tube.

T. due

0.19 0.21 0.24 0.26 0.29 0.32 0.36 0.39 0.43 0.48 0.52 0.57 0.62 0.68 0.74 0.80 0.86 0.93 1.00 1.07 1.14 1.21 1.28 1.35 1.42 1.49 1.56

-I- T

6.70 6.75 6.80 6.85 6.90 6.95 7.00 7.05 7.10 7.15 7.20 7.25 7.36 7.35 7.40 7.45 7.50 7.55 7.66 7.65 7.70 7.75 7.80 7.85 7.90 7.95 8.00

/

,

,

,

-

E. olkalr

24.81 24.79 24.76 24.74 24.71 24.68 24.64 24.61 24.57 24.52 24.48 24.43 24.38 24.32 24.26 24.20 24.14 24.07 24.00 23.93 13.86 23.79 23.72 13.65 13.58 ,3.51 z3.44

6.70 6.75 6.80 6.85 6.90 6.95 7.00 7.05 7.10 7.15 7.20 7.25 7.30 7.35 7.40 7.45 7.50 7.55 7.60 7.65 7.70 7.75 7.80 7.85 7.90 7.95 3.00

- c

- -

cc. due cc. acid

2.31 22.69 2.29 22.71 2.26 22.74 2.24 22.76 2.21 22.79 2.18 22.82 2.14 22.86 2.11 22.89 2.07 22.93 2.02 22.98 1.98 23.02 1.93 23.07 1.88 23.12 1.82 23.18 1.76 23.24 1.70 23.30 1.64 23.36 1.57 23.43 1.50 23.50 1.43 23.57 1.36 23.64 1.29 23.71 ! .22 23.78 1.15 23.85 1.08 23.92 ..Ol 23.99 I.94 24.06

cc. dye

0.25 0.28 0.31 0.34 0.38 0.42 0.46 0.50 0.55 0.60 0.65 0.71 0.77 0.84 0.90 0.97 1.04 1.11 1.18 1.25 1.32 1.39 1.46 1.53 1.60 1.67 1.73

c. ozkaz

24.75 24.72 24.69 24.66 24.62 24.58 24.54 24.50 24.45 24.40 24.35 24.29 24.23 24.16 24.10 24.03 23.96 23.89 23.82 23.75 23.68 23.61 23.54 23.47 23.40 23.33 23.27

EC. dye cc. acid

2.25 22.75 2.22 22.78 2.19 22.81 2.16 22.84 2.12 22.88 2.08 22.92 2.04 22.96 2.00 23.00 1.95 23.05 1.90 23.10 1.85 23.15 1.79 23.21 1.73 23.27 1.66 23.34 1.60 23.40 1.53 23.47 1.46 23.54 1.39 23.61 1.32 23.68 1.25 23.75 1.18 23.82 1 .ll 23.89 1.04 23.96 1.97 24.03 1.90 24.10 1.83 24.17 I.77 24.23

consistency with the above equations. This consistency, as stated above, has been previously demonstrated by Gillespie, but the possibility of using this method to estimate pH values closer than 0.2 intervals has not before been emphasized.

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698 Calorimetric Plasma pH

Determination of the pH of Plasma or Serum.

The discussion below covers the technique used with 0.2 cc. samples of plasma and 0.4 cc. samples of whole blood. The only changes necessary when larger samples are taken are the use of

6.6 _. . .

6.7 i ,, , , / c+mllfaeq 1

6.8 \I \t I I I : Dztm nbcd

7.2

g 73

7.4

15

7.6

7.7

73

7.9

7.0

7.1

8.0

CcohQnolredinl (I25 0.35 Q4S a56 a65 Q75 Q% 095 105 16 12!5 1.35 14 155

Cc&et& redin I 225 215 2A5 195 185 1.75 1B 1.55 1.45 W 125 115 105 095

FIG. 1. The solid lines are drawn through the points satisfying the

equations pH,oo = 7.78 + log alkaline form

acid form and ~Hw = 7.65 + log

alkaline form

acid form respectively. The X’s represent the readings actually

obtained.

proportionally larger volumes of saline solution for dilution, and larger test-tubes.

4 cc. of adjusted saline indicator solution are pipetted under oil into a small test-tube (1.5 X 10 cm.). After addition of 0.2

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A. B. Hastings and J. Sendroy, Jr. 699

cc. of serum or plasma, the mixture is gently stirred with a footed glass rod. A control tube is prepared in a similar manner, using saline solution without dye. The oil is replaced by paraffin, which

TABLE II.

Values of pK’ for Phenol Red Calculated at 20” and 58”.

BA HA pH200

ob- served.

EC. dye cc. dye

1.50 1.00 7.96 1.45 1.05 7.92 1.40 1 .lO 7.89 1.35 1.15 7.85 1.30 1.20 7.79 1.25 1.25 7.77 1.20 1.30 7.74 1.15 1.35 7.72 1.10 1.40 7.68 1.05 1.45 7.64 1.00 1.50 7.61 0.95 1.55 7.56 0.90 1.60 7.52 0.85 1.65 7.50 0.80 1.70 7.46 0.75 1.75 7.41 0.70 1.80 7.37 0.65 1.85 7.33 0.60 1.90 7.28 0.55 1.95 7.23 0.50 2.00 7.17 0.45 2.05 7.11 0.40 2.10 7.05 0.35 2.15 7.00 0.30 2.20 6.93 0.25 2.25 6.84

Mean.

~K’20’ dye

calcu- lated.

7.78 7.78 7.78 7.78 7.75 7.77 7.78 7.79 7.78 7.78 7.79 7.77 7.77 7.79 7.79 7.78 7.78 7.78 7.78 7.78 7.77 7.77 7.77 7.79 7.80 7.79

7.78

~K’20” clY?

‘“Y’IE r mean.

*o.oo

*o.oo

=+=o.oo

*o.oo

-0.03 -0.01 *o.oo +0.01 *o.oo *O.OO $0.01 -0.01 -0.01 +0.01 $0.01 *o.oo *o.oo *0.00 =+=O.OO *o.oo -0.01 -0.01 -0.01 $0.01 $0.02 t-o.01

-0.0004

3H380 G’38a ,b8er- calcu- ved. lated.

PK’38. hivi~

mean.

7.81 7.63 -0.02 7.78 7.64 -0.01 7.76 7.65 *O.OO 7.72 7.65 *o.ca 7.66 7.62 -0.03 7.64 7.64 -0.01 7.60 7.64 -0.01 7.58 7.65 *0.00 7.55 7.65 *o.oo 7.51 7.65 *o.oo 7.49 7.67 +0.02 7.44 7.65 AO.00 7.39 7.64 -0.01 7.37 7.66 $0.01 7.33 7.66 $0.01 7.28 7.65 *o.oo 7.24 7.65 *o.oo 7.20 7.65 10.00 7.14 7.64 -0.01 7.09 7.64 -0.01 7.04 7.64 -0.01 6.99 7.65 10.00 6.91 7.63 -0.02 6.87 7.66 $0.01 6.81 7.68 +0.03

7.65 -0.0024

7.96 7.92 7.89 7.85 7.82 7.78 7.74 7.71 7.68 7.64 7.60 7.57 7.53 7.49 7.45 7.41 7.37 7.33 7.28 7.23 7.18 7.12 7.06 6.99 6.91 6.83

- ’ i .

- j . 1 _

/

,

,

,

-

7.83 7.79 7.76 7.72 7.69 7.65 7.61 7.58 7.55 7.51 7.47 7.44 7.40 7.36 7.32 7.28 7.24 7.20 7.15 7.10 7.05 G.99 6.93 6.86 6.78 6.70

is allowed to solidify, and the tube of unknown pH is brought to a temperature lo to 0.5” higher than required (usually to 39” or 38.5’), by immersion in a water bath. It is then placed in the comparator block, to be read when the temperature has fallen

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700 Calorimetric Plasma pH

to 38”. The block should have three rows of holes to admit tubes placed between the eye and the source of light in the manner indi- cated in Fig. 2. The known group, consisting of an alkaline tube, acid tube, and control tube is then compared with the unknown group, consisting of two water tubes and the unknown tube. By careful comparison, using standards at 0.05 pH intervals, one may interpolate as accurately as when but two tubes are used with buffer stand- ards; i.e., to 0.02 pH. 5 cc. portions of the bicolor standards prepared as above are placed in the tubes used for the comparison with unknowns. The above amounts of sample and saline are one-fifth of the amounts used by Cullen.

Litjilht I - I

COntrOt tclb4 - lciiol

- Utiertube

Acid tubQ - ba - Lhter tube

Alkaline tibQ -----+ a(q - Unknown tube

t EYQ

FIG. 2. Arrangement of tubes in comparator for reading calorimetric pH of blood by means of the bicolor standards.

Determination of the pH of Plasma in a Sample of Whole Blood.

This is essentially the technique proposed by Hawkins (7). Instead of 0.2 cc. of serum, 0.4 cc. of whole blood is taken as a sample and added to adjusted saline, the indicator content of which has been corrected as described in the next paragraph, for the increased volume of approximately 0.2 cc. due to the cells. The mixture is stirred and centrifuged, and the pH is determined as above. In this determination, it is necessary that the mineral oil be replaced by paraffin before centrifugation.

Saline Indicator Solution.

This solution, which contains per liter 0.154 M NaCl and 0.0000222 M phenol red, is prepared as follows: 0.9 gm. of NaCl is dissolved in freshly redistilled water in a 100 cc. flask. 10.50 cc. of 0.0075 per cent phenol red solution are added if the saline is to be

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A. B. Hastings and J. Sendroy, Jr. 701

used in plasma determinations, 11.00 cc. if it is to be used for whole blood. The mixture is then diluted to the 100 cc. mark. A 0.154 M solution of NaCl without phenol red is prepared at the same time for control tubes.

The saline is adjusted to the approximate blood reaction as follows: At the beginning of each experiment, after addition of the phenol red, as indicated above, the solution is covered with paraffin oil. 0.01 N NaOH is admitted by capillary pipette, with stirring until approximately pH 7.4 is reached. Saline adjusted in this manner is hardly changed by standing overnight.

Control of Temperature.

Since the temperature has no effect on the colors of the alkaline and acid standard solutions, one needs only to control the tem- perature of the tube containing the serum. This may be simply done by immersing the tube in a beaker and gently heating until the desired temperature is reached. It is essential that a ther- mometer, held in place by a stopper so that it does not touch the sides of the tube, be used to determine the exact temperature of the solution. When the unknown solution is covered with solid paraffin, its temperature may be obtained by using the ther- mometer on either the control, or one of the water tubes, which has had exactly the same treatment as the unknown.

EXPERIMENTAL.

E#ect of NaCl on Bicolor Standards.

Since in the determination of its pH, serum is diluted with ,0.154 M NaCl (0.9 per centj the effect of salt in this concentration on the bicolor standards was determined. This was done by making up two sets of bicolor standards, one in water and the other in 0.154 M NaCl. The concentrations of indicator, alkali, and acid were those used in the previous experiments. The tubes were matched against phosphate standards having the same amount of dye. The results of this experiment, given in Table III, indicate that in the presence of 0.154 M NaCl the bicolor standards are 0.01 to 0.02 pH more acid in appearance

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702 Calorimetric Plasma pH

than when no salt is present. A more complete study of the effect of salts in varying ionic strengths on the activity of the dye is in progress.

Determination of the pK’ of Phenol Red.

Table II indicates how this value, at 20” and 38”, was obtained. The pH values of bicolor standards made up in water were deter- mined by comparison with phosphate standards. By substitut- ing the value of the pH observed in Equation I, pK’ was calculated for each pair of bicolor tubes. The values 7.78 at 20’ and 7.65 at 38” represent the mean of all such calculations of the pK’ of the dye at the two temperatures.

TABLE III.

Ej’ect of 0.164 M NaCl on the Bicolor Standards.

Temperature.

20”

38O

- - pH of standard.

N&l absent. NaCl present.

7.38 7.36 7.45 7.44 7.53 7.52 7.59 7.61

7.14 7.14 7.24 7.23 7.33 7.30 7.39 7.36

Average............................................

salt “error.”

0.02 0.01 0.01 0.02

0.00 0.01 0.03 0.03

0.016

As an incidental result of the previous experiment on the effect of NaCl, one may calculate the pK’ of phenol red in the absence of salt. Due to the effect of the phosphate on the activity of the salt of phenol red in the alkaline tubes, the color of the phosphate tube corresponded to that of a solution more acid by 0.02 than the real PH. Therefore, under conditions of zero salt the pKfzoo of phenol red would be 7.76. Similarly, the pKfs80 would be 7.63.

Comparison of Macro and Micro Determinations.

The desirability of using as little blood as possible for the determination *of pH led Hawkins to the demonstration that

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A. B. Hastings and J. Sendroy, Jr.

Cullen’s method could be modified and successfully used on 0.25 cc. of blood. Several comparisons between the Cullen and Hawkins’ methods have been made in this laboratory with satis-

TABLE IV.

Comparison of pH Determinations of Serum Made by Phosphate Standards and Bicolor Standards on 0.1 Cc. Samples.

NO. pH readings.

Phosphate standards. 1 Bicolor standards.

1 7.41 7.42 2 7.42 7.42 3 7.30 7.29 4 7.32 7.33

TABLE V.

Effect of Standing on Bicolor Standards.

pH on June 2, 1924. pH on June 10.1924. Change in pH.

7.96 7.05 -0.01 7.92 7.92 *o.oo 7.89 i.90 +0.01 7.85 7.88 to.03 7.79 7.79 *o.oo 7.77 7.77 *o.oo 7.74 7.74 to.00 7.72 7.72 *o.oo 7.GS 7.68 *o.oo 764 7.64 *o.oo 7.61 7.62 $0 01 7.56 7.56 +o 00 7 50 7.49 -0.01 7.46 7.47 $0.01 7.41 7.43 +0.02 7.17 7.17 +o.oo 7.11 i.l? +0.01 6 93 6 94 +0.01

- -.

factory agreement between the two methods. To determine whether the bicolor standards could be used as successfully in the micro method as in the macro method, pH determinations of serum made with phosphate standards and those made with

1 When pH determinations are made in the presence of cells, as in the Hawkins method, it is imperative that readings be made within half an hour after centrifugation, otherwise errors due to acid formation will occur.

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704 Calorimetric Plasma pH

bicolor standards in small test-tubes were compared. The results of a few such comparisons given in Table IV show satisfactory agreement between the two methods.

Stability of Bicolor Standards.

Although standard tubes of phosphate and phenol red are not reliable for use in blood pH determinations after 3 days, we have found that the bicolor standards remain practically unchanged after 8 days (Table V). The maximum length of time for which the standards may be regarded as reliable is as yet undetermined.

Comparison of Electrometric and Calorimetric pH Values at 38’.

A series of experiments was made in which the pH values of plasma from the blood of various species, at different CO2 tensions, were determined both electrometrically and calorimetrically at 38”. These results are given in Table VI. The species studied were man, horse, dog, and rat. In all, twenty-three such experi- ments have been performed with the result that the average difference between the determinations made in the two different ways is -0.003 with a maximum difference of ~0.04. The differences encountered are not outside the combined errors of the two methods and it may therefore be concluded that, within the limits of experimental accuracy, the calorimetric pH determina- tions by Cullen’s method when read at 38’ are identical with electrometric pH determinations. That this identity is real and not accidental is shown by the fact that the plasma of two species, the dog and rat, which gave at room temperature such widely different Cullen corrections as 0.35 and 0.12, respectively, show no differences between their calorimetric and electrometric values at the same temperature.

Standard Phosphates at 58’.

In order to read calorimetric pH values at 38” it was necessary to have the standard pH solutions at the same temperature as the unknown, otherwise the question of the effect of temperature on the dye would be involved. The pH of standard phosphate solutions made up as described by Cullen was therefore deter- mined electrometrically at 38”, using as the standard of reference

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A. B. Hastings and J. Sendroy, Jr.

TABLE VI.

705

Compan’son of Electrometric and Calorimetric pH Values.

Date.

1984

Apr. 9

“ 29

Man.

Horse.

‘( 24 Dog.

“ 11 Rat.

‘I 10

May 28

Horse.

“

.June 3 ‘I

Jan. 9

Apr. 8

Horse.

Man.

-

--

_-

_-

-

No.

1 2 1 2 3 4 1 2 3 4 1 2 3 1 2 1 2 3 4 1 2 3 4

1 2 3 1 2 3 4

“~z- 38”.

7.34 7.53 7.31 7.46 7.32 7.46 7.25 7.40 7.26 7.40 7.01 7.20 7.35 7.22 7.50 7.52 7.50 7.35 7.33 7.39 7.39 7.21 7.22

Electro- metric

200.

7.27 7.46 7.72 7.15 7.37 7.56 7.62

-

--

_-

-

-

Colori- metrio

339.

7.33 7.53 7.33 7.47 7.34 7.47 7.27 7.41 7.27 7.40 6.99 7.16 7.35 7.26 7.51 7.52 7.49 7.36 7.34 7.40 7.41 7.23 7.25

Colori- metric

204.

7.26 7.46 7.69 7.14 7.36 7.57 7.60

Average deviation............................... Maximum “ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

,H, - pH

+0.01 *o.oo -0.02 -0.01 -0.02 -0.01 -0.02 -0.01 -0.01 10.00 +0.02 +0.04 10.00 -0.04 -0.01 *0.00 +0.01 -0.01 -0.01 -0.01 -0.02 -0.02 -0.03

to.01 10.00 to.03 to.01 to.01 -0.01 to.02

-0.003 hO.04

-

c

_ _

,

.-

- t

-

-

Cullen. “ “ “ “ “ “ ‘I “ “

Hawkins. “ “

Cullen. “ ‘I “ “ “

Bicolor. “ “ “

Cullen. “ “ “ I‘ I‘ “

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706 Calorimetric Plasma pH

TABLE VII.

Electrometric pH Values at .%?OO” and 38’ of aa/16 Phosphate Mixtures. Average Results of Two Experiments.

MI15 NaaHPO4

cc. cc.

49.6 50.4 52.5 47.5 55.4 44.6 58.2 41.8 61.1 38.9 63.9 36.1 66.6 33.4 69.2 30.8 72.0 28.0 74.4 25.6 76.8 23.2 78.9 21.1 80.8 19.2 82.5 17.5 84.1 15.9 85.7 14.3 87.0 13.0 88.2 11.8 89.4 10.6 90.5 9.5 91.5 8.5 92.3 7.7 93.2 6.8 93.8 6.2 94.7 5.3

6.809 6.781 -0.028 $0.001

6.862 6.829 -0.033 -0.004 6.909 6.885 -0.024 $0.005 6.958 6.924 -0.034 -0.005 7.005 6.979 -0.026 $0.003 7.057 7.028 -0.029 *O.OOO 7.103 7.076 -0.027 +0.002 7.154 7.128 -0.026 +0.003 7.212 7.181 -0.031 -0.002. 7.261 7.230 -0.031 -0.002 7.313 7.288 -0.025 +0.004 7.364 7.338 -0.026 +0.003 7.412 7.384 -0.028 +0.001 7.462 7.439 -0.023 +0.006 7.504 7.481 -0.023 $0.006 7.561 7.530 -0.031 -0.002 7.610 7.576 -0.034 -0.005 7.655 7.626 -0.029 *O.OOO 7.705 7.672 -0.033 -0.004 7.754 7.726 -0.028 +0.001 7.806 7.776 -0.030 -0.001 7.848 7.825 -0.023 +O.OOS 7.969 7.877 -0.032 -0.003 7.948 7.919 -0.029 *O.OOO 8.018 7.977 -0.041 -0.012

Mean difference.. _._ Average deviation from mean difference.. .

-0.029

TABLE VIII.

Error Involved in Failure to Adjust Saline.

P 20 -38” Deviation

from mean difference.

+o .00004

Electrometric pHzoo. Calorimetric pH3oo. Dilution = 21 times.

Saline adjusted. I Saline unadjusted.

7.27 7.26 7.24 7.46 7.46 7.45 7.72 7.69 7.66

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A. B. Hastings and J. Sendroy, Jr. 707

the B of the saturated calomel cell as determined with 0.1 N HCl and assuming as its pH 1.08, at 38’. Two complete series at 0.05 pH intervals were run. The results of these determinations are given in Table VII, which gives the ratio of Na2HP04 and KHzPOa corresponding to 0.05 pH intervals from pH 6.8 to 8.0. A comparison of these values with those obtained at 20’ shows

the latter to be 0.03 more alkaline at any given NazHPO, KH po ratio.

2 4

Similar results have been obtained by Cullen.

Adjustment of Reaction of Saline Solution Used for Diluting Hood.

Cullen has emphasized the necessity of adjusting to 7.4 the pH of saline solution used in diluting. the serum. The results of experiments given in Table VIII demonstrate the necessity of

TABLE IX.

Comparison of Mineral Oil and Parafin in Preventing Loss of CO,.

200 Sample.

Paraffin........................... la Oil.. . . . . . . . . . . b

Paraffin. . . . Oil. . . . . . . . .

Paraffin. . . . 3a Oil. . . . . . b

Paraffin. Oil .

. .

.

2a 7.62 7.67 +0.05 b 7.61 7.64 +0.03

4a b

Date Date June 3, June 4,

1824. 1924. pH change overnight.

7.63 7.63 10.00 7.62 7.66 +0.04

7.41 7.41

7.43 7.44

7.41 *o.oo 7.48 +0.07

7.44 +0.01 7.50 +0.06

such adjustment. They show that when the pH of the serum is near that of the saline, the calorimetric pH at 21-fold dilution is within 0.01 to 0.03 of the electrometric pH at 20”, whereas the pH of the solution of serum in unadjusted saline is 0.02 to 0.06 less at the same dilution. At higher dilutions the discrepancy becomes greater.

Use of Parafin to Cover Solutions.

CulIen originally recommended the use of oil to cover the solu- tions whose pH was to be determined. Hawkins prepared tubes

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708 Calorimetric Plasma pH

of such dimensions that they were entirely filled up to the stopper with solution. We have.found that it is an advantage to replace the oil with paraffin after the serum or blood has been mixed with saline solution. Loss of COz by passage from the solution into the oil introduces an error which, although not significant at room temperature, seems to be of measurable magnitude at 38”. Table IX shows the effect of allowing the tubes to stand overnight when covered with paraffin and with oil. As might be expected, the greater increase in alkalinity with standing is in those tubes with a higher initial COz tension.

DISCUSSION.

Factors Involved in the Cullen Calorimetric Correction.

The Salt Error.-Among the factors composing the calorimetric correction, the salt error of the dye might have been conceived to play a r61e. This is negligible in the determinations as carried out by Cullen’s method. It will be shown in a later paper that the so called salt error of phenol red is really a change in the activity of the ions which is a function of the ionic strength of the solutions (Lewis and Randall, 8). The ionic strength of the saline solution is 0.154 p and of the phosphate standards, 0.134 p at G.8 and 0.190 p at 7.8. Thus the ionic strengths of the standard and unknown solutions arc approximately the same and any effect produced on the activity of the dissociated dye will be the same in both solutions.

The Protein Error.-Although the influence of proteins on the color of phenol red is great in undiluted serum or plasma, at the 21-fold dilution which Cullen chose as the optimum dilution for reading calorimetric serum pH, this error is negligible.

The Dilution Error.-It is, of course, conceivable that diluting a solution with a non-buffered NaCl solution will lead to a pH which is different from that of the original undiluted solution: i.e., there may be an error in the resulting pH introduced by dilution. This possibility we have tested in the following manner.

Solutions, 30 my with respect to NaHC03 and 100 mM with respect to NaCl, were equilibrated with CO, at 38”. After dilution ranging from O- to al-fold with adjusted, isotonic N&l solution, the pH of each solution was determined calorimetrically at 20” and 38”. The results of this experi- ment are given in Table X. The pH was not significantly changed by dilution in any case.

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A. B. Hastings and J. Sendroy, Jr. 709

One may conclude, therefore, that al-fold dilution, per se, with an isohydrionic, isotonic NaCl solution has no measurable effect upon the pH of the solution.

Effect of Temperature.

Since the system contains not only BHCOI and H&03 but also proteins and their salts, variations of the temperature effect from that of a simple BHCOrH2C03 solution are to be expected. In plasma we have from this standpoint the following equilibria

H&OS = & + HCOa + 2,800 cal. (Landolt-Bknstein, 9)

HP = G + ‘; + 10,000 cal. approximately (Meyerhof, 10)

TABLE X.

Eflect of Dilution with Isotonic NaCl on the pH of Bicarbonate Solutions at .90° and 38’ C.

Solution.

_

-

Dilution.

0 5

10 15 21

0 5

10 15 21

I _-

-

200

7.65 7.60 0.05 7.64 7.66 0.04 7.64 7.60 0.04 7.65 7.60 0.05 7.64 7.59 0.05

7.68 7.63 0.05 7.66 7.62 0.06 7.66 7.61 0.05 7.66 7.61 0.05 7.65 7.60 0.05

38” Difference.

According to the van? Hoff isochore the change in pK’~&o~ from 38” to 20” should be +0.12 and in pK’HP, +0.43. The difference in the pH of plasma at 38” and at 20’ would therefore lie somewhere between 0.12 and 0.43, with the occurrence of variations in this difference depending on the character and con- centration of the serum proteins. The temperature effects ob- served by Cullen are within these limits. The variations in the

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Calorimetric Plasma pH

effects with species and pathological condition are comprehensible, as well as the desirability of avoiding such variations by per- forming the determinations at body temperature.

SUMMARY.

Using pairs of tubes to give varying ratios of the alkaline and acid forms of the indicator, it has been demonstrated that phenol red in bufferless solutions may be successfully used in place of phosphate standards, for the estimation of the reaction of blood or serum to within f 0.02 pH.

It has been found that calorimetric and electrometric deter- minations of the pH made on the same sample of blood agree to within 0.02 pH when both determinations are made at the same temperature. The correction used by Cullen for calorimetric determinations performed at room temperature is unnecessary when the readings are made with the diluted plasma at body temperature.

BIBLIOGRAPHY.

1. Cullen, G. E., J. Biol. Chem., 1922, lii, 501. 2. Bjerrum, N., Die Theorie der alkalimetrischen und aaidimetrischen

Titrierungen, Stuttgart, 1914. 3. Myers, V. C., J. Biol. Chem., 1922, liv, 675. 4. Gillespie, L. J., J. Am. Chem. Sot., 1920, xlii, 742. 5. Michaelis, L., Die Wasserstoffionenkonzentration, Berlin, 2nd edition,

1922. 6. Clark, W. M., The determination of hydrogen ions, Baltimore, 1922. 7. Hawkins, J. A., J. Biol. Chem., 1923, lvii, 493. 8. Lewis, G. N., and Randall, M., Thermodynamics, New York and

London, 1923. 9. Landolt, H., Barnstein, R., and Roth, W. A., Physikalisch-chemische

Tabellen, Berlin, 4th edition, 1912. 10. Meyerhof, O., Arch. ges. Physiol., 1922, cxcv, 53.

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A. Baird Hastings and Julius Sendroy, Jr.WITHOUT BUFFER STANDARDS

BLOOD pH AT BODY TEMPERATURE OFCOLORIMETRIC DETERMINATION

STUDIES OF ACIDOSIS: XX. THE

1924, 61:695-710.J. Biol. Chem. 

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