a new colorimetric method for the estima

8/6/2019 A New Colorimetric Method for the Estima

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A NEW COLORIMETRIC METHOD FOR THE ESTIMA-TION OF POTASSIUMBY H. R. D. JACOBS* AND WILLIAM S. HOFFMANi

(From the Department of Medicine, Rush Medical College, the University ojChicago, Chicago)(Received for publication, August 12, 1931)

The usual method for the estimation of potassium in the bloodand the urine depends, as s well known, upon the precipitation ofthe element as the cobalti-nitrite and the subsequent estimationof one of the constituents of the precipitate. The reducing powerof the precipitate is used by Kramer and Tisdall(l); the formationof a diazo color by the nitrite is employed by Doisy and Bell (2);the potassium is titrated as the acid tartrate by Fiske and Litarc-zek (3); the cobalt is determined as the thiocyanate by Breh andGaebler (4).

From a theoretical point of view those methods which determinedirectly a stable constituent of the precipitate are preferable tothose which depend upon the activity of the unstable nitrite rad-ical. The methods of Fiske and Litarczek and of Breh and Gaeb-ler embody this principle, but each of these has a disadvantage:that of Fiske and Litarczek involves an ashing and a second-pre-cipitation, and that of Breh and Gaebler depends upon a foreignfactor (alcohol concentration) as well as upon the factor being esti-mated.It is the object of this paper to describe the formation of a newstable, colored compound of cobalt and its application to the microestimation of potassium in biological fluids.

MethodPrinciple-When a solution of sodium ferrocyanide is added toa mixture of a cobaltous salt and choline hydrochloride in water* Ernest E. Irons Fellow in Medicine; under the supervision of Dr.

George F. Dick.t National Research Council Fellow in Medicine; under the supervisionof Dr. Wilber E. Post.085

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686 Calorimetric Estimation of Potassiumsolution, an emerald-green color develops immediately. Prelim-inary experiments with this color showed that it was sensitive tominute amounts of cobalt, choline, and ferrocyanide, and that,in the presence of a given excess of choline and ferrocyanide, itcould be used to determine calorimetrically small variations in theconcentration of cobalt. It was further established that the col-ored solution might be diluted with water without affecting itsquality, and that it developed to maximum intensity in a fewminutes and maintained this intensity for hours.Procedure-The precipitation of potassium from blood serumis carried out according to the method of Ifiamer and Tisdall.Into a 15 cc. conical centrifuge tube marked at 6 cc. is pipetted 1cc. of serum. 2 cc. of Kramer and Tisdalls sodium cobalti-nitritereagent are added slowly with constant agitation of the serum.After 45 minutes, 2 cc. of water are added, the contents mixed, andthe tube centrifuged at moderate speed for 15 minutes.The tube is then inverted and drained briefly. 1 cc. of water isrun down the side of the tube, the tube centrifuged 5 minutes, andagain inverted and drained. The precipitate is then suspended in2 cc. of 70 per cent alcohol with the aid of.a stirring rod, the tubecentrifuged 5 minutes, inverted, and drained. The alcohol wash-ing is repeated once.After the tube has drained for a few minutes, the precipitate issuspended in 1 cc. of water with the aid of a stirring rod. Anothercc. of water is added and the tube then immersed in a boiling waterbath for 10 minutes. The contents of the tube are agitated severaltimes during the heating in the bath. The precipitate will dissolveto form a clear, colorless solution. A small amount of insolublematerial, probably protein, does not interfere. When the tubehas cooled, 1 cc. of 1 per cent choline hydrochloride and 1 cc. of 2per cent sodium ferrocyanide are added to the solution and madeup to the 6 cc. mark.1 Sodium Cobalti-Nitrite Reagent-Solution A: 25 gm. of cobaltous nitratecrystals are dissolved in 50 cc. of water and to this solution are added 12.5cc. of glacial acetic acid. Solution B: 120 gm. of sodium nitrite (potassium-free) are dissolved in 180 cc. of water. This gives a total volume of about220 cc. To all o f Solution A are added 210 cc. of Solution B. An evolutionof nitric oxide gas occurs at once. Air is drawn through the solution untilall the gas has passed off . The reagent (thus prepared it has a pH of 5.7)is placed in the ice chest and filtered each time before using. It will keepat least 1 month.


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H. R. D. Jacobs and W. S. Hoffman 687The precipitation of potassium from ashed or inorganic solutions

is made from 2 cc. of the unknown solution with 1 cc. of thereagent. The first washing with water after centrifugation isomitted, and the precipitate washed twice with 70 per cent alcoholas outlined for serum. The remainder of the procedure is thesame.

The colored solution thus formed is compared with a standardcolored solution made from 1 cc. of standard cobalt nitrate (orsulfate), 1 cc. of 1 per cent choline hydrochloride, and 1 cc. of 2 percent sodium ferrocyanide made up to 6 cc. with water. Thestandard cobalt solution is made from either the nitrate or the sul-fate: 0.6701 gm. of Co(N0&.6H20 or 0.6469 gm. of CoS04.7Hz0made up to 1000 cc. with water. These solutions must be stand-ardized because these cobalt salts are hygroscopic. The stand-ardization is made against the cobalt contained in the precipitatesof potassium from a standard potassium sulfate solution contain-ing 0.4011 gm. of KzSO~ per liter (equivalent to 18 mg. of .K per100 cc.).

CommentThis method embodies the theoretical advantage of determining

directly a stable constituent of the potassium precipitate. Theprocedure is relatively simple. The use of a permanent standardpermits of more accurate comparisons between values obtainedat different times, and serves as a check upon the efficiency of theprecipitating procedure when this is in question.

There are only two accurate measurements to be made in eachdet.ermination: that of measuring 1 cc. of the serum to be usedand that of diluting the colored solution to the 6 cc. mark. Allof the manipulations are carried out in the original tube so thatthere is no loss from transfers.

Washing the precipitate in alcohol has the advantage that it isthoroughly freed from the precipitating agent without danger ofloss, since it is insoluble in alcohol. The alcohol also makes theprecipitate cohere well so that the supernatant fluid may bepoured off and the tube drained upside down. It was found thatthere was a large amount of cobalt in the first water washing, asmall amount in the first alcohol washing, a trace in the secondalcohol washing, and none in the combined third alcohol washings


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688 Calorimetric Estimation of Potassiumfrom eight tubes in a test experiment. When the precipitatesare washed with water according to the method of Kramer andTisdall, even the fourth washings contain perceptible amounts ofcobalt, which must come either from the residual precipitatingreagent or from solution of the precipit.ate itself.

The quantity of cobaltous salt to be used in the preparation ofthe standard cobalt solution is calculated from the formula of theprecipitate, &NaCo(NO& .H*O, in which the amount of potas-sium is to be 0.18 mg. (equivalent to that in 1 cc. of solution con-taining 18 mg. per 100 cc.). Cobaltous nitrate and sulfate rapidlyabsorb water during exposure to air, so that weighing is not accu-rate, and standardization against a known potassium solution isnecessary. Once made, the cobalt solution keeps indefinit,ely.If the sulfate is used for the standard cobalt solution, it may alsobe standardized by determining the sulfate in the solution.

The choline solution is made from choline hydrochloride crystals.A stock solution of 10 per cent, from which the 1 per cent solutionis made by dilution, may be kept in the refrigerator. Dry crystalsmay be conveniently prepared by precipitating an alcoholic solu-tion with ether.

The sodium ferrocyanide is made in 2 per cent solution fromNalFe(CN)6. 10HzO. It is preserved in a cool, dark place. Whenit becomes very yellow or when it develops a red precipitate, itshould be discarded. Of course, potassium ferrocyanide may beused instead.

The quantities of choline and ferrocyanide used in this methodare sufficient for any amounts of cobalt encountered in potassiumdeterminations. In developing the color, the choline solutionshould be added to the cobalt solution first, and then the ferro-cyanide. If the ferrocyanide is added to the cobalt solutionbefore the choline, the color develops much more slowly, insteadof immediately, as in the given procedure.

The object in adding 1 cc. of water as the first washing after thecentrifugation is to remove as much of the protein-containing fluidas possible before washing the precipitate with alcohol. Thepresence of too much coagulated protein makes disintegration ofthe precipitate somewhat more difficult.

If the precipitate is dissolved as outlined above, solution will berapid and complete. The small particles which sometimes remain


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H. R. D. Jacobs and W. S. Hoffman 689are probably protein, but they give up their salt readily andbecome translucent flakes which cause no difficulty. They settle

TABLE IDeterminations of Potassium in Known Potassium SolutionsTT K found K taken K found

w7. WI.0.16 0.16660.16 0.16000.18 0.17860.18 0.18300.20 0.20*0.20 0.20120.24 0.24200.24 0.2400

K takenI II

WI. w7. m!J.0.10 0.1043 0.09910.10 0.1064 0.09990.11 0.1100 0.10980.11 0.11* 0.10910.12 0.1204 0.12*0.12 0.1204 0.12070.14 0.1433 0.14760.14 0.1386 0.1363

--

-* The solution against which the other solution s of the series were com-pared.

TABLE IIDeterminations of Potas sium in Human Blood Sera*Totassium Potsssium -

_ .

-

-

_-

-

KtTTisdrtllmethod

Authorsmethodafterashing

m7.per cent17.1

21.720.0

Case No.

6789

10

KEYTisdallmethod

Authorsmethod

pzent18.618.719.419.721.921.922.322.120.620.9

m 7 .per cent19.919.918.421.121.321.421.421.321.3

mg .per cent17.018.519.618.2

19.920.2

mg .per cent19.719.9

?w.per cent16.619.419.019.920.220.719.8

- -* Ten consecutive cases.

out after the colored solution is formed and remain in the tubewhen the solution is poured into the calorimeter cup.


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690 Calorimetric Estimation of PotassiumColor comparisons are best made by daylight. The colors areeasily matched in any good calorimeter.Tables I to IV present the results of determinations carriedout by this method upon several types of solutions. Tables IIIand IV show that known amounts of potassium added to urineand serum can be determined accurately.

TABLE IIIDeterminations of Potassium in Urine with Added PotassiumK in urine K added Total K K found

mp. per cent mg. per cent mg. per cent mg. per cent178.1 24.0 202.1 204.1178.1 48.0 226.1 226.2178.1 72.0 250.1 257.6178.1 96.0 274.1 277.4

The known amounts of potassium were added to measured portions ofthe urine, the mixtures then ashed, and the potassium determined in analiquot of the solutions of the ashes. Two cobalt standards, one equivalentto 18 mg. of K per 100 cc., and the other to 36 mg. of K per 100 cc., were used.

TABLE IVDeterminations of Potassium in Serum with Added Potassium

K in serum

peR!ePcnt18.618.618.618.6

Solution AK added Tota l K~-

fw .pm cent peznt1.8 20.43.6 22.25.4 24.07.2 25.8

K found K in serum K added Tota l K--WI.per cent p2Lt peznt ma.pm cent

19.9 22.2 1.8 24.022.2 22.2 1.8 24.024.1 22.2 3.6 25.825.9 22.2 5.4 27.622.2 7.2 29.4

K foundml.per cent

24.023.825.927.529.8

Ammonium salts interfere in this determination because am-monia also forms a very insoluble cobalti-nitrite precipitate.For this reason glassware should be cleaned shortly before theprecipitation is made, and, for this reason also, fluids, such asurine, which contains appreciable amounts of ammonia, mustfirst be ashed.In serum potassium determinations it must be remembered that


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H. R. D. Jacobs and W. S. Hoffman 691the red corpuscles contain relatively much more potassium thanthe serum, and that potassium values will be incorrect in hemo-globin-tinged serum and in serum which has been in contactwith the clot for a long time. It is best to collect the blood inperfectly dry (preferably baked) glassware, allow it to clot in anincubator for 15 minutes, and then centrifuge it, and separate theserum at once.

SUMMARY1. The formation of a hitherto unmentioned colored solutionfrom cobalt, choline, and ferrocyanide is described.2. A method is outlined for the determination of potassium inblood, urine, and ash solutions by means of this color.3. Data on determinations of potassium in blood sera, in urine,and in inorganic solution are presented.

BIBLIOGRAPHY1. Kramer, B., and Tisdall, F. F., J. Biol. Chem., 48, 339 (1921).2. See Briggs, A. P., J. Biol. Chem., 67,351 (1923).3. Fiske, C. H., and Litarczek, G., J. Biol. Chem., 87, p. xvi (1926).4. Breh, F., and Gaebler, 0. H., J. Biol. Chem., 87, 81 (1930).


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a new colorimetric method for the estima

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