Estimation of the Sulfonamides A Rapid and Accurate Micromethod S. W. LEE, N. B. HANNAY,
AND
W. C. HAND
Wallace Laboratories, Inc., New- Brunswick, 4 . J.
R
E C E N T work in the sulfonamide field, in which attempts are being made to correlate low blood ievels with efficacy, has made exact determinations of these drugs more important than ever. This need, and the desire to obtain the advantages of working on a micro scale, have led to a method which has most of the advantages of tlie methods now in wide use, and practically none of their shortcomings.
T.iBLE
I. FREESULFATHIAZOLELEVELS
Subject and Sulfathiazole Dose
Regular B r a t ton and Marshall Method
.rrQ.% Rabbit, 0.5 gram orally, blood taken after one hour Man, 2 grams taken orally, blood taken after 2 hours Man, 1 gram taken orally, blood taken after 2 hours Rabbit, 0.5 gram taken orally, sample after 1 hour M a n 2 grams orally. sample after 1 hour X a n , 2 grams orally, sample after 4 hours
llicromettiod Blood laked Blood pptd. before pptn. directly IIU. % -!Iff. %
3 . 0 (trip.)
......
3 . 5 (trig.)
2 , 9 (dupl.)
......
3 . 6 (dupl.)
2.8
3 . 3 (dupl.)
3 . 3 (dupl.)
3 . 5 (quad.)
4.0 (dupl.)
4.0 (dupl.)
2 , 3 (quad.)
2 , 6 (quad.)
2 . 6 (trip.)
2 . 7 (trip.)
3 1 (quad.)
3 . 1 (quad.)
The methods most coninionly used are those of Bratton and Marshall (2) and Werner (8), or modifications or adaptations of them. I n the former procedure, the sulfonamide is diazotized, and the diazonium salt coupled n-ith 5(1 -naphthy1)ethylenediamine dihydrochloride. The azo dye which is formed is determined colorimetrically. The undesirable features of this procedure have been mentioned in a preliminary note on the method under discussion ( 5 ) . I n the Kerner method, p-dimethylaminobenzaldehydeis used to form a yellow ani1 with the “free” sulfonamides, and the intensity of this color is measured. It is difficult to use this method for accurate micro work because of the l o x tinctorial value of the yellow dye. It has also been pointed out that “total” values obtained n-ith these methods are subject t o error resul cing from the change in color intensity with small changes in p H
furic acids (“acid mixture”). A small amount of sulfuric acid was found to aid materially in a quick and complete precipitation of the protein, and was present in sufficient amount for hydrolysis purposes, for use in the “total” sulfonamide determinations. Laking the blood prior to precipitation is unnecessary (Table I). The precipitated protein is filtered off, the diazotization is carried out, ethyl alcohol is added, and the naphthylethylenediamine dihydrochloride is added immediately. The color attains its maximum intensity in 15 seconds. I n the presence of alcohol, it is not necessary to destroy the excess nitrous acid, for possible products formed by its reaction with the dye :ire likely to be of the same color and soluble in the medium. S o nitrogen hubbles are formed, because the sulfamatenitrous acid reaction is eliminated, and little or no interference from bubbles (7) was noticed. Parallel experiments 4 i o ~ e dthat the sulfamate addition is not necessary, the colors being even more stable in its absence. The recoveries obtained by different means are shown in Tables I, IV, and V. In the tables, results are expressed in different terms for the sake of clearness. In some cases milligram per cent figures are given; in others, actual density readings from the drum of the Coleman spectrophotometer are given. Density is defined as the iogarithm of the reciprocal of the fraction of transmitted light. If Beer’s law is obeyed, as it is in this case, the density is a linear function of the concentration. All results have been checked. Elapsed time was measured with a stop watch. All blood mas added at the ratio 1 to 20. All blood samples were whole and oxalated, and usually less than 0.5 hour old. It was found that blood which contained sulfathiazole increased in apparent sulfa drug content by about 10 per cent after standing at room temperature for 20 hours. Appropriate concentrations of trichloroacetic acid were present in all diazotizations. All density readings were made against reagent blanks of the same age. Accurate analysis was possible for a period of 24 houi>. The stability of the colors in the various methods is shon n in Table 11. TOTAL SULFADRUGS. I n the determination of total sulfa drug concentration, the Bratton and Marshall method is much to be preferred to the shorter method of Kerner (x), primarily because of the negligible effect of mineral acids on tlie intensities of the azo colors formed. I n the micromethod 0.1 ml. of 4 N sulfuric acid is used in the hydrolysis, although 0.4 ml. of the acid does not seriously alter the density r e d -
(6).
The time required for a single analysis is greatly reduced using the micromethod described below; an analysis may be completed in 7 or 8 minutes. This is even less than the time necessary for the Kerner method, which is about 12 minutes (1). Reasonable amounts of sodium chloride do not interfere. Fifty mg. per cent or less of potassium thiocyanate (6) did not interfere in the determination of 10 mg. per cent of sulfathiazole by the Bratton and 1Iarshall or the micromethod. This concentration of thiocyanate is much higher than is ever obtained in the blood. Blank readings on normal blood (human, horse, and rabbit) were found to be zero as a rule, and never exceeded 0.2 mg. per cent.
TABLE11.
Method Aqueous sulfathiazole solution, Bratton and Marshall procedure Aqueous sulfathiazole solution, micromethod without sulfuric acid Aqueous sulfathiazole solution, micromethod Sulfath a r d e in rabbit blood filtrate, micromethod
Determination of Sulfa Drugs
FREESULFA DRUGS. I n the micromethod, tlie blood is precipitated directly in a mixture of tricliloroacetic and sul403
COLORS FORMED IN VARIOVS METHODS
STaBILITY OF
Density Reading of a 10-up. Sample of Sulfathiazole after: 1 2 3 4 2 4 hour hours hours hours hours
5 min.
0.280.25
0.20
..
.,
0.15
0.26
0.25
...
0.255
0.26
0.26
0.25
...
0.28
0.26
0.26 0.25
0.25
0.245
...
,.
..
0,235
INDUSTRIAL AND ENGINEERING CHEMISTRY
404
TABLE111. RECOVERIES OF SULFATHIAZOLE ADDEDTO URINE Final Dilution
Recovered by Bratton and hlarshall
Recovered by Micromethod
Q
%
90 92 96 99
90 98 99 100
1:10 1:20
1:3O 1:60
TABLEIV. RECOVERIES OF SULFATHIAZOLE FROM HORSE BLOOD (Re ular procedures as outlined were followed. Sulfathiazole was added t t a t each 2-ml. aliquot of filtrate should contain 2 , 4 , 6 , 8 , or 10 mg. of the drug.)
EO
2 mg.
%
Sulfathiazole Recovered 4 mg. 6 mg. 8 mg.
%
1.85 1.86
3:$6
5.8
7:$5
9.5
1.95
4.0
5.8
7.84
9.6
added
Bratton and Marshdl Micromethod Micromethod without sulfuric acid
%
10 mg.
%
Method
added added added Milliuran per cent 5.27
%
added 8.5
ings. If the acid mixture of trichloroacetic and sulfuric acids is used for precipitation of the blood, the filtrate may be used directly for the total sulfa drug determination. SULFADRUGSIN URINE. The recovery of sulfathiazole added to various dilutions of normal urine is shown in Table 111, compared with results obtained on the same samples by the Bratton and Marshall procedure.
Recovery of Sulfa Drugs from Whole Blood
Vol. 15, No. 6
added to 2.00 ml. of solution, both in the reaction with and without added sulfuric acid. The indicated presence of sodium chloride means that 1.0 ml. of 0.7 per cent saline mas added. The experimental conditions for diazotizations at 35' C. were the same as those in Table IV. The addition of a small amount of sulfuric acid to the usual trichloroacetic acid decreased the recovery very slightly. The recovery of sulfathiazole added to whole blood, using the micromethod, is almost quantitative (95 to 100 per cent). A comparison of recoveries is given in Table IV. The variation in recoveries among the sulfa drugs has been explained on a solubility basis: the more insoluble the sulfonamide, the greater the error in the determination due to loss in preparing the blood filtrate (4). This explanation seems unlikely when one considers that only micrograms are present in solutions in which tenths or hundredths of grams will dissolve, so that solutions are often one ten-thousandth suturated. The sulfa drugs vary in solubility by factors of 10 or 20. Solubility would thus be expected t o exert little influence on the recoveries. Adsorption of the drug on the precipitated protein can play no important role in producing the low results, for complete (98 to 100 per cent) recovery can be obtained by the micromethod after blood has been precipitated in the presence of the sulfa drug a t 1 to 20 dilution. The Bratton and Marshall procedure gives 85 per cent recoveries under these conditions. On the blood of subjects who received sulfathiazole orally, the micromethod gives results that are about 15 per cent higher than those given by the Bratton and Marshall method. This was taken to indicate complete recovery by the modified method, in conjunction with the results given in Table V. Table I gives typical blood analyses by the two procedures, and shows that laking the blood before precipitation is not necessary.
Much has been written (7) about the recovery of sulfa drugs from blood. It is the general opinion that, using the Bratton and Marshall method, added sulfathiazole is recovered from whole blood to the extent of 85 to 90 per cent, a t the dilution of 1 to 20. This was confirmed, as shown in Tables IV and V. Work TABLEV. RECOVERIES OF SULFATHIAZOLE ADDEDTO VARIOUSMEDIA previously reported (6) and experiments carried Diazotization SulfathiaSulfathiazole out in this laboratory indicate that the filtrates Tempersole Added Found from precipitated blood (with added sulfathiazole Method Time ature Medium 1 2 1 2 and a t 1 to 20 dilution) contain practically all the Min. e c. hfQ. % hfQ. % B. & M. 3 ca.20 Waterand CClaCOnH 4 8 3.2 6.0 sulfathiazole (95 to 100 per cent). Low recoveries 3 ca.20 Water and CClsCO4 8 4.0 8.0 B. & M. were obtained with the Bratton and Marshall (in presence of NaCl) OH B.&M. 10 ca.25 WaterandCClaCOZH 4 8 4.0 8.0 procedure when the sulfathiazole was added to 3 25 Rabbit blood before 4 8 3.5 6.4 B. & M. precipitation the whole blood, when whole blood was laked and B. & M. 3 25 Sterile, oxalated horse 5 6 3.6 3.7 precipitated in the presence of added sulfathiazole, blood before laking, etc. and in some cases when the sulfathiazole was Micro 3 25 Sterile, oxalated horse 5 5 4.9 4.9 blood before laking, added to blood filtrates (Tables I, IV, and V). etc. This is contrary, in part, to the findings of B. & M. 3 25 Filtrate from rabbit 4 8 3.6 6.6 blood Sunderman and Pepper ( 7 ) . 4 25 B. & M. Filtrate from human 4 8 3.8 7.1 blood These results indicate that the ratesof diazotiza33. & hl. 3 ca. 22 Trichloroacetic acid 10 10 8 8 tion in various media are of importance. The soln. before addn. and ptn. of rabbit diazotization experiments are summarized in bloox B. & M. 10 ca. 22 Trichloroacetic acid 10 10 9.8 9.9 Tables VI and VII, which give rates and extents s o h . before addn. of the various reactions. These experiments and ptn. of rabbit blooa indicate that the variation in recoveries with B. & M. 3 25 Trichloroacetic acid 4 8 3.7 6.5 s o h . before addn. different drugs, and with different conditions of and ptn. of rabbit precipitation (medium, temperature, dilution), is bloog B. & M. 3 25 Trichloroacetic acid 10 10 9.9 10 to be explained on the basis of incompleteness (in presence of NaCI) soln. before addn. and ptn. of rabbit of the reactions leading to the formation of bloog the dye. 3.7 Trichloroacetic acid 4 8 B. & M. 3 25 6.6 The diazotizations at 2 5 O C. summarized in Table VI were carried out in a constant-temperature bath, using the procedure indicated. In the Bratton and Marshall reactions 0.10 ml. of freshly prepared 0.100 per cent sodium nitrite was added t o 3.00 ml. of solution. In the micromethod the same quantity of nitrite was
B. & hI.
3
25
Micro
3
25
soln. before addn. and pptn. of rabbit blood Sterile, oxalated horse blood laked in distilled water Sterile,oxalatedhorse blood laked in distilled water
5
5
3.5
3.6
5
5
4.9
4.9
ANALYTICAL EDITION
June 15, 1943
These results indicate that the low recoveries obtained by the Bratton and Marshall method may be due in some cases to incomplete diazotization. The rate of diazotization is influenced b y the temperature, sodium chloride and other catalysts, mineral acid content, and possibly by retarding substances in the blood filtrates. If the routine time for diazotization is to be 3 minutes, careful note must be made of these conditions. Higher recoveries (by the Bratton and Marshall method) were frequently obtained when diazotization was carried out for longer than 3 minutes, or when the temperature was markedly higher than 25” C. Addition of sodium chloride to one of the reagents in the regular Bratton and hIarshal1 procedure would preclude the chance of incomplete diazotization, and reduce the time for this step to 1 minute. Cooper, Gross, and Hogan (3) recommend diluting the blood with normal saline. Using this method they obtained high recoveries, which they attributed to the prevention of hemolysis on diluting the blood. It might be possible that they are, in part, due to the catalytic effect of the salt on the diazotization.
TABLE
1‘1. R.4TES
OF
DIAZOTIZ.4TION O F SULFATHIAZOLE VARIOUS MEDIA
Medium and Method Aqueous sulfathiazole solution, B. & hl. procedure Raobit blood filtrate, B. & Af. procedure Aqueous sulfathiazole solution, added NaC1, B. & AI. procedure Aqueous sulfathiazole solution, micromethod Rabbit blood filtrate, micromethod Rabbit blood filtrate, micromethod without sulfuric acid
Per Cent Reaction after Diazotizing for: 1 2 3 5 min. min. rnin. min. rnin. At 2 5 0 0,5
c.
46
62
87
90
100
58
79
92
95
100
96
100
100
100
100
83
100
100
100
100
83
100
100
100
100
83
96
97
100
100
At 350 Aqueous sulfathiazole solution, B. & M. procedure Rabbit blood filtrate, B. & &I. procedure Aqueous sulfathiazole solution, micromethod Aqueous sulfathiazole solution, micromethod, without sulfuric acid
IN
405
TABLE VII. RATESOF DIAZOTIZATION OF SEVERALSULFA DRUGSIX VARIOUSMEDIA 0;5 Medium and .Method Aqueous sulfacetamide solution, B. & 51. procedure Aqueous sulfanilamide solution, B. & M. procedure Aqueous sulfapyridine solution, B. & 31. procedure Aqueous sulfacetamide solution, micromethod Aqueous sulfanilamide solution, micromethod Aqueous sulfapyridine solution, micromethod Sulfathiazole in 2 ml. of distilled water containing CChCO2H and 1 mi. of alcohol
Per 1 Cent 2Diazotized 3
5
min.
min.
min.
rnin.
min.
100
100
100
100
100
46
62
79
92
96
58
75
92
96
100
100
100
100
100
100
79
98
100
100
100
100
100
100
100
100
...
...
...
50
...
in 10 ml. flat-bottomed vials. These were found to be convenient, since they do not require racks, and allow complete mixing of the reagents by shaking. To each of a series of vials were added 0.47 ml. of water, 0.43 ml. of 15 per cent trichloroacetic acid, 0.10 ml. of 4 N sulfuric acid, and 1.00 ml. of water or one of the various concentrations of sulfathiazole solutions. The procedure gave solutions comparable to 2 ml. of blood filtrate. The sulfathiazole solutions were made up as follows: 0.1000 gram of c. P. sulfathiazole was dissolved in 1 liter of distilled water. Various amounts of this solution (2, 4, 6, 8, and 10 ml.) were diluted to 100 ml. The resulting dilutions contained 2, 4, 6, 8, and 10 micrograms of sulfathiazole per ml. The color was developed as in the micromethod, using 0.10 ml. of nitrite, waiting 3 minutes, and then adding 1.00 ml. of alcohol and 0.10 ml. of Bratton and Marshall’s reagent. By plotting the densities (or the per cent transmission) of the colors against the concentration (2, 4, 6, 8, and 10 micrograms per determination), a very useful calibration curve was obtained. If the outlined procedure for blood is followed, the same scale (2,4,6,8, and 10) will correspond to the milligram er cent of sulfathiazole in the sample taken. In other words, miligram per cent is the same as micrograms per 0.1 ml. Beer’s law is exactly obeyed to a concentration of at least 12 micrograms per determination.
c.
67
83
100
100
100
83
100
100
100
100
100
100
100
100
100
93
100
100
100
100
Experimental Procedure REAGEXTS.Trichloroacetic acid, 3.33 per cent. Trichloroacetic and sulfuric acid (“acid mixture”). Sulfuric acid (56 ml. of 4 N) is added to 1 liter of 3.33 per cent trichloroacetic acid. Sodium nitrite. An aqueous solution of c. P. sodium nitrite (0.1 per cent) is used. This was found to be very stable (more than a month in summer weather). It was renewed when low readings were obtained from known amounts of sulfa drugs. Bratton and Marshall’s reagent. N(1-naphthy1)ethylenediamine dihydrochloride (0.1 per cent in water) was used. It was found possible to use this for a matter of months also, providing the determinations are made against reagent blanks. Ethyl alcohol, undenatured 95 per cent ethanol. INSTRUMENT. All determinations were made with a Coleman Universal s ectrophotometer, with the wave length dial set at 550 mp. A! colors were compared against reagent blanks of the same age, in microcuvettes of 2.5-ml. volume. The similarity in the absorption curves of the dyes formed in the two methods would indicate the method to be equally useful with other colorimetric instruments. CALIBRATION CURVE. Calibration curves were run in distilled water containing the concentration of acids which were present in the regular analyses. All the final colors were formed
Procedure for Blood and Urine PRECIPITATION OF THE BLOOD. Whole blood (0.30 ml.) is added dropwise to 5.70 cc. of trichloroacetic acid, or to the same volume of “acid mixture”. Each drop of blood is broken up by vigorous stirring with the end of the pipet, and the tube is agitated by hand after all the blood is added. The mixture is allowed to stand until the precipitated protein settles and is then filtered through either No. 1 or 42 Whatman paper of about 6-cm. diameter. The filtrates are uniformly water-clear. Slightly more than 4 ml. are collected, sufficient for the determination of both “free” and “total” sulfonamides. DETERMINATION OF FREESULFADRUGIN WHOLEBLOOD. Exactly 2 ml. of the filtrate (from either precipitating acid) are pipetted into 10 ml. flat-bottomed vials, sodium nitrite (0.10 ml. of 0.1 per cent) is added, and at least 3 minutes are allowed for the diazotization. Alcohol (1.00 ml.) is added, and the tube is swirled. On mixing Bratton and Marshall’s reagent (0.10 ml.) with the solution, the characteristic color is produced. The formation of the color is complete within 15 seconds, and the densities of the solutions are reasonably constant for 24 hours. Reading in all cases should be made against reagent blanks, and values obtained from a calibration chart. DETERMISATION O F TOT.& SULFA DRUGSI N WHOLE BLOOD. Exactly 2 ml. of the filtrate from the “acid mixture” precipitation, or 2 ml. from the trichloroacetic acid precipitation with 0.10 ml. of 4 N sulfuric acid added, are heated in a boiling water bath for about one hour. The volume is adjusted to 2.00 ml. with distilled water, and the procedure for the free determination is followed. DETERMINATION OF SULFADRUGSIN URINE. Urine (0.10, 0.20, or 0;30 ml.) is added to the mixed acid blood-precipitating reagent (5.90, 5.80, or 5.70 ml., respectively), representing dilutions of 1 t o 60, 1 to 30, or 1 to 20. The pipet is rinsed with the solution. The Sam le is filtered if any turbidity develops. The solution or a t r a t e 6.00 ml.) is analyzed according to the method
406
Vol. 15, No. 6
INDUSTRIAL AND ENGINEERING CHEMISTRY
outlined for blood. The colors formed in the presence of diluted urine are stable for only an hour, with both Bratton and Marshall's and the micromethods.
and hIarsha11 procedure. The micromethod has proved yery reliable and useful for large-scale work,
Literature Cited
Summary A micromethod for the estimation of sulfonam'des is based on the coupling of completely diazotized sulfonamides to N(1-naphthy1)ethylenediamine. Changes in the procedure have removed most of the sources of objection to the Bratton and Marshall method. The time for a single complete analysis has been reduced to about 8 minutes. The colors formed are stable for 24 hours. Recovery of sulfathiazole added to whole blood has been shown to be essentially complete at a dilution of 1 to 20. On blood from subjects receiving the drug orally, the micromethod gives results which are uniformly about 15 per cent higher than those obtained by the Bratton
Andrews, M. C., and Strauss, A. F., J . Lab. Clin. M e d , 26, 888 (1941). (3)
Bratton, A. C., Marshall, E. K., Jr., Babbitt, Dorothea, and Hendrickson, A. R., J . BioE. Chem., 128, 637 (1939). Cooper, F. B., Gross, Paul, and Hogan, M. L., Am. J.Clin. Path.,
(4)
Hoffman, W. S.,"Photelometric Clinical Chemistry",
(2)
12, 149 (1942). p. 224,
Yew York, William Morrow and Co., 1941. (5) Lee, 9. W., Hannay, N. B., and Hand, W. C., Science, 97, 359 (1943).
Morris, C. J. O., Biochem. J., 35,952 (1941). (7) Sunderman, W. F., and Pepper, D. S., Am. J . Med. Sci.. 200, 792 (1940) ( 8 ) Werner, A . E. A , Lancet, 1, 18 (1939).
Microdetermination of Mercury in Organic Compounds H. WILLIAM ECKERT Division of Laboratories and Research, New York State Department of Health, Albany,
D
ESPITE the desirability of a quantitative method for
the determination of mercury in biologic products that have been preserved with phenyl mercuric acetate, Merthiolate, or other organic mercury compounds, no entirely satisfactory micromethod has been published. I n the author's experience the Gettler and Lehman modification (a) of the Winkler method (6, 7) for the determination of mercuric salts, although satisfactory for mercuric ions, has given very low results with organic mercury compounds such as those mentioned, except under certain circumstances when phenyl mercuric acetate may be titrated lb-ith dithizone as a monovalent ion. The Gettler and Lehman method consists, briefly, of a nitric acid and permanganate digestion followed by destruction of excess permanganate by nitrite, removal of excess nitrous acid by hydroxylamine sulfate, and titration of t h e metal ion by dithizone.
N. Y.
Standardization of the titration technique also contributes greatly to the accuracy of the determination, as described below. Under the conditions described, no evidence of oxidation of the dithizone has been encountered. APPARATUS.Pyrex reflux apparatus consisting of a 50- or 100-ml. round-bottomed flask and Liebig-type condenser with a 25-em. outside water-cooled jacket, REAGENTS.Dithiaone Solution. Dissolve 12.5 to 13.0 mg. of diphenylthiocarbazone (Eastman) in 500 ml. of carbon tetrachloride (Baker, analyzed, in bottles), and allow to stand for a day in the dark. It is important t o use the best carbon tetrachloride obtainable. Filter through paper and store in a dark brown bottle in the dark. The titration value of this reagent remains constant for at least a month. Hydroxylamine Sulfate Solution. Dissolve 20 grams of hydrovylamine sulfate (Eastman) in 100 ml. of water.
Experimental Since the losses mentioned above might be due either to incomplete digestion or to volatilization, many modifications of the digestion conditions were made in an attempt to avoid these errors. Two methods gave satisfactory results: Carius digestion in a microbomb and treatment of the material with aluminum in a neutral or slightly alkaline medium at 75' to 80" C. The disadvantages of the first were the difficulty in manipulation and the formation of chlorine oxides which had to be removed. The reaction with aluminum provided a practical digestion method and its application to the determination of organic mercurials is the subject of this report. The reaction between metallic aluminum and the organic mercurials studied ran smoothly and quantitatively at 75' to 80" C.a t an initial p H range between 7.8 and 8.4. I n order to obtain consistent end points in the dithizone titration, a certain minimum concentration of salts in the aqueous phase is essential. Amounts exceeding the minimum have no detrimental effect. Varying the concentration of acids over the range studied has no effect on the titration (Table I).
T.4BLE
I.
TITRATION O F MERCURY WITH DITHIZONE
S'ARIATIONS I N
mercury in the presence of different concentrations of acids and salts) Amounts of Salts and Concentrated Dithizone in Acids in 100 Ml. of Solution Titrations Average (0.109 mg.
'W.
M1.
M1.
MI.
500 mg. of Tyrode's saltsa 0 . 5 ml. of Hzs0.1 11.4 11.3 11.3 11.3 0 . 5 m l , o f H ~ S O ~ + 1 m l . o f H N O a 11.4 11.4 11.3 11.4 0.5rnl.ofHzSOif2ml.ofHNOa 11.2 1 1 . 2 . 11.3 11.2 11.4 11.3 0 . 5 ml. of HsS04 3 ml. of HNOa 11.3 11.3 11.2 11.2 0 . 5 m l . o f H ~ S O ~ + 5 m l . o f H N O a 11.3 11.2 No added salts 9.9 9.9 0 , 5 ml. of His04 10.2 9.7 0.5ml.ofHzS0~+1ml.ofHNOa 9.4 9.9 10.0 9.8 9.9 9.9 0.5ml,ofHzSO~+2ml.ofHXOa 9.7 10.1 0 . 5 m l . of HzSO4 3 ml. of HNOa 10.3 10.0 9.7 10.0 10.3 10.3 0 . 5 ml. of HzS04 5 ml. of HNOs 9.5 11.2 0 . 5 mi. of H?SO4 5 ml. of HNOs 11.3 11.3 0 . 5 gram of NaCl 11.3 11.4 11.2 11.3 1 . 0 gram of NsCl 11.4 11.3 2 , 0 grams of NaCl 11.2 11.3 11.4 11.3 Composition of Tyrode's salts ( 4 ) : NaCl 8.0, KC1 0.2, CaClz 0.2, MgClr 0.1, NaHzPOh 0.05, AaHCOa 1.0, and d-glucose 1.0 gram.
+
+
++
