New hydrogen electrodes and rapid methods of determining hydrogen ion concentrations
J. F. McClendon
From the Physiological Laboratory of the University of Minnesota
Received for publication May 8, 1915
The technique of hydrogen electrodes as applied to pure chemistry is in a high state of perfection1 but the impression is sometimes given that these electrodes require a skilled physical chemist to use them correctly. It is true that some solutions give trouble, for example, a pure KCl solution. Starting with Merck's highest purity KCl, I recrystallized it five times in fused silica dishes and dissolved it in conductivity water, but could not obtain a neutral reading with the hydrogen electrode until I poured it boiling hot into the electrode and passed a rapid stream of hydrogen through it while cooling and while making the reading. This difficulty is never experienced with solutions containing considerable amounts of carbonates, phosphates or proteins, and hence biological fluids are less difficult than some inorganic solutions. In fact, it is not necessary with biological fluids to change the hydrogen in the electrode during the reading.
The time required to determine the reaction of a fluid depends chiefly on the time required to saturate the electrode with hydrogen. Since gold absorbs comparatively little hydrogen, it becomes quickly saturated. I found No. 36 gold wire very satisfactory from this standpoint but the electrodes made of it could not be so conveniently cleaned by heating, as platinum electrodes. Drucker made electrodes of films of iridium burned on Jena glass, but the same objection holds true of them. By reducing the thickness of the platinum, the saturation time may be reduced. I found that platinum foil 0.02 mm. in thickness, coated with platinum black, requires less than two minutes for saturation, provided it is separated from the hydrogen by only a film of the solution. Since the electrodes which Michaelis designed for rapid work require thirty minutes for saturation2 a considerable saving of time is thus accomplished by simply using narrow strips of thin foil instead of the wire that he used.
The chief difficulty in determining the H+ concentration of biological fluids arises from the fact that they contain dissolved gases. The dilution of the hydrogen with other gases causes an error in the direction of greater acidity. The loss of C02 from the solution increases its alkalinity, hence the passage of C02 and 02 from the solution into the hydrogen causes two errors which tend to oppose one another, and hence the reading might happen to be correct. Hober, Hasselbalch, Michaelis and others have guarded against the error due to escape of C02 from the solution. The method of Michaelis depends on the use of a very small volume of H2 in ratio to the volume of the solution, and is best adapted to rapid work.
The chief difficulty with other gases is experienced in determinations on arterial blood. The oxyhemoglobin gives out so much O2 into the H2 as to cause a greater error than arises from the escape of CO2. Milroy3 centrifuged the blood and then poured it through the air into the electrode. In order to obviate errors arising from this procedure, as well as other errors, and to shorten the time required for a determination, I designed the following electrode:
This electrode consists essentially of a U-tube with one end constricted, figure 1. Next to the constriction, a strip of platinum foil, 0.02 mm. in thickness or thinner, is fused through the glass so that the plane of that portion of the foil which protrudes into the interior of the tube passes through the axis of the tube. The free end of the foil, A, is bent up so that when a loop of wire is passed over the constricted end of the tube electric contact will be made with the foil. A short piece of rubber tube, B, is attached to the constricted end of the glass tube, and closed with a small Langenbeck clip or pinch cock at C. The rubber tube is as short and of as small bore as practicable, and its free end is connected with a hypodermic needle. The large end of a hypodermic needle is filed down so that it may be inserted into the rubber tube. The platinum is coated with platinum black and cleaned in the usual manner.
Before filling the electrode, the needle is dried thoroughly be means of a suction pump and filled with oil. It may then be boiled for sterilization. The rubber tube is filled with a concentrated solution of hirudin in water or Ringer. The needle is inserted into the artery or vein and the Langenbeck clip removed. The U-tube is held in such a position that only the first few drops of blood come in contact with air, as this first blood covers and protects the rest of the blood. When the U-tube is filled, the Langenbeck clip is put on the rubber tube and the needle is removed.
The U-tube is now placed in the shield of a centrifuge, counterbalanced, and centrifuged a few minutes. It is then removed and a pipette inserted into the free end of the rubber tube. By sucking on the pipette and at the same time partially opening the Langenbeck clip, the blood corpuscles that remain in the rubber tube are removed. A tube from which pure hydrogen is flowing is instantly put in place of the pipette, without admitting any air. By cautiously opening the Langenbeck clip, hydrogen is admitted until the platinum foil is surrounded by the gas. A film of plasma adheres to the platinum and sides of the glass tube and establishes electrical connection with the blood below. After waiting two minutes for the platinum to be saturated with H2, the U-tube is shaken so as to bring a fresh portion of the plasma in contact with the platinum, and the reading is immediately taken. Care should be taken not to shake so hard that any of the corpuscles rise high enough to liberate any oxygen into the hydrogen. In order that no time be lost in connecting this electrode with the calomel electrode, a ball of cotton cord soaked in a saturated solution of KCl is kept at hand and a piece cut off previously, with which to make the connection. The inclosure of this conducting cord in a tube is unnecessary.
Since the reading can be made in a few seconds with a proper potentiometer, the C02 does not have time to diffuse out of the film of plasma covering the platinum and cause an error. Since the erythrocytes have all been precipitated away from the surface layers, and the exposure is but little more than two minutes, the contamination of the hydrogen with other gases is very slight, and the error due to this unmeasurable.
This electrode has been indispensable in my work on blood, but it can be used with any biological fluid. In studying stomach or duodenal contents, the rubber tube may be connected to the smallest size stomach or duodenal tube, having a strainer (bucket) on the end that
is swallowed. The filling of the U-tube may be assisted by aspiration through a rubber tube attached to its free end, but the suction should not be sufficient to cause bubbles to appear in the fluid.
In case the readings are made at room temperature, it will be necessary to insert the U-tube in mercury in order to cool it with sufficient rapidity. If blood or any other fluid that requires to be centrifuged is used, the centrifuge shield may be filled with water so that most of the cooling takes place without loss of time.
If extreme accuracy is not required and the fluids to be investigated, contain no oxyhemoglobin, the electrode designed by Michaelis may be used, provided thin platinum foil is substituted, and a bulb is blown on the open end of the glass tube to prevent spilling while introducing the H2. A strip of foil 0.05 mm. thick may be drawn through a small rubber stopper by means of a needle and thread, instead of sealing it in a glass stopper. This foil may be cleaned by heating, provided the stopper is first wet with distilled water, whereas a glass stopper is liable to crack.
In measuring the acidity of the gastric contents, it. was found possible to lower an electrode into the stomach. The apparatus designed for work on the stomach contents consists chiefly of a rubber tube 60 cm. long and 3 mm. bore, and two No. 40 silk covered copper wires, that were coated with rubber cement and dried several times (fig. 2). One wire, M, extends through the rubber tube, JJ, and the other, JV, passes down outside of it until by entering the hole, E, it connects with a platinum wire that is fused into the lower end of a short piece of glass tube that is inserted into the rubber tube. The lower end of the glass tube and copper-platinum junction is covered with sealing wax, A. A drop of pure mercury is dropped into the lower end of the glass tube so as to connect with the platinum wire at the level of B. Above the mercury a little calomel washed with concentrated KCl solution, C, is placed, and the rest of the glass tube packed with moist KCl crystals, D, and the hole, E, stuffed with cotton soaked in KCl solution. This forms a calomel electrode, and is separated off from the remainder of the tube by a short piece of glass rod, F. Above F several holes are cut in the rubber tube at the level of G, and from this point a fine platinized platinum wire extends through the lumen of the tube and is held in place by fusion to a bump on the inside of a short piece of glass tube at the level of I. This platinum wire then connects with the wire M and the junction is coated with rubber. The rubber tube is connected at К with a tube, L, leading from a hydrogen generator, and a slow stream of H2 passes down the rubber tube and out at G, thus converting the platinum wire from H to F into a hydrogen electrode. Whereas the results are not quite as accurate as those obtained with larger electrodes, I think them sufficiently accurate for stomach contents, where there are such great individual variations. It is necessary to have a source of hydrogen of sufficient pressure to prevent the stomach contents from rising in the tube higher than Я. Fresh crystals of KC1 must be put in D before the apparatus is used, and the end of the tube from A to G may be immersed in saturated KCl solution so as to keep it moist until it is swallowed. A correction is made for hydrogen pressure (subtract 0.17 mv. for 1 cm. increase in pressure).
The time necessary to calculate the H+ concentration from the potentiometer reading may be saved either by using a conversion table or making a potentiometer that reads off directly the H+ concentration. It is most convenient to express results in the form of the hydrogen ion exponent, PH. Thus 0.001 normal is 10-3, or pH = 3. Figure 3 is a conversion table to be used at 23°, provided the hydrogen electrode is connected with a calomel electrode of the saturated type (containing KC1 crystals). The coordinates are to be followed only to the edge of the diagonal band. Starting with the potentiometer reading in millivolts on the ordinate, the corresponding PH on the abscissa may be read.
The temperature coefficient is small and is practically zero for an H+ concentration of 0.0001 normal (PH = 4). That is to say a 0.0001 normal solution of hydrogen ions will give a reading of 481 millivolts whether the temperature is 19°, 25° or 37°. If 23° is taken as the standard, at 19° there is a variation of +3 millivolts for PH = 0 and -5 mv. for PH = 10. At 37° the variation is -11 mv. for PH = 0 and +16 mv. for PH = 10.
Since the PH of gastric juice is not far on the acid side of 4, that of urine is about 4 and that of the other biological fluids (except those containing much bile) is not far on the alkaline side of 4, a slight variation of the temperature from 23° does not make a serious error in using this conversion table or a potentiometer reading the PH directly.
None of the potentiometers on the market can be easily adapted to read the PH directly, but I made one that will serve all such purposes. It consists of 2200 cm. of No. 30 hardened German silver wire stretched over cross section paper. By means of a Weston cell, and 2 movable contacts, the current of a lead storage cell passing through a variable length of the wire is so adjusted that each centimeter has a fall of potential of one millivolt. By means of a conversion table the PH is marked on the cross section paper. We thus have a scale reading the PH instead of the millivolts. See also the description of a direct reading potentiometer in this journal.
The method of H+ concentration as used by Michaelis is modified so as to reduce the time necessary for a determination from forty minutes to about two or three minutes.
An electrode which eliminates errors due to 02 (from oxyhemoglobin) and C02, is described. An electrode that may be lowered into the stomach is also described.
A potentiometer reading the H+ concentration directly, instead of millivolts, is described.