ANALYTICAL CHEMISTRY
808,
Under favorable conditions a single estimation may be completed within 110 minutes of which 40 minutes would be Kitrogen, Kitratepersonal-attention time. This compares P P.M. Coefficient Fiducial Limits KO.of (.\lean Standard of Variafavorably with 3 5 hours (with 40 minutes Sample Replicates Yalue) Deviation tiona 5% 1% Phalaris 14-7 8 1160 16 1 39 1147-1173 1140-1180 personal-attention time) for the phenol Phalaris 26-6 15 2517 16 0 62 2508-2526 2505-2529 disulfonic acid method ( 7 ) with samples Wild oats 27-6 a 8896 41 0 46 8862-8930 8845-8947 of high nitrate content involving small 0 Standard Deviation X 100 aliquots and minimal evaporations on the Mean water bath; with samples of lower nitrate content larger aliquots would be required and longer evaporations would solution but is rapid in acid solution. To ensure the complete abincrease the time of operation. With the method described here sorption of nitrate ions, it is thus essential that the reaction of the a large number of estimations may be carried out concurrently extract from the plant material be definitely acid, and that the by setting up a series of resin columns. acidity be provided by an acid of lower activity ton ards the resin Further, the proposed method requires a smaller number of reathan nitric acid; in fact the further down the list the better. gents than does the phenol disulfonic acid method, and is therefore Kere it not for the fact that chloride interferes with the final subject to less chance of reagent blank. It also avoids the use of estimation of nitrate, dilute hydrochloric acid would probably ammonia which is an important consideration in a laboratory such provide a very satisfactory medium for the extraction of nitrate as this where a large number of Kjeldahl determinations are carand its subsequent complete retention by the resin. The next conried out. venient choice falls to acetic acid, as plant extracts with this acid The preposed procedure is thus a high-precision, short-time are very easily filtered and the end point of the silver chloride premethod capable of being used for routine field investigations as well cipitation is readily observable. For some reason, as yet unas for precise laboratory research work. known, recoveries were not so complete when acetic acid was used. Phosphoric acid of 1% v./v. strength was finally chosen as an ACKNOWLEDGMENT extracting medium. When it was used the recoveries of nitrate The authors wish to acknowledge gratefully the guidance and added to P. tuberosa extracts were complete, and analytical results interest of H. R. Marston, F.R.S., Chief of the Division of Biowere very consistent, although phosphoric acid extracts from the chemistry and General Nutrition, C.S.I.R.O. They also wish to grasses do not filter quite as rapidly as those made with acetic thank C. S. Piper for his helpful discussions in the development and acid, and furthermore the end point of the removal of chloride by modifications of the methods employed, and A. E. Cornish for his silver sulfate is slightly obscured by the precipitation of silver phosadvice in the statistical interpretations. phate. But neither of these objections is serious. Sodium hydroxide was chosen for the elution. . 4 strength of LITERATURE CITED 4% w./v. is necessary so that 50 ml. of the solution will neutralize Barnes, H., Analyst, 75, 358 (1950). 50 ml. of 1% phosphoric acid and provide sufficient excess for reBlom, Jacob, and Treschow, Cecil, 2. Pfianz., Dung U . Bodenk., generation. 13A, 159 (1929). A yellow color frequently appears in the eluate. The pigment Eastowe, J. E., and Pollard, A. G., J. Sci. Food Agric., 1, 266 in plant tissues which is responsible is probably held by adsorption (1950). Egner, H., Ericksson, E., and Emanuelsson, A., Ann. Roy. rather than by exchange with the resin. However, its presence is Agric. Coll. Sueden, 16,593 (1949). no disadvantage subsequently, provided the solution is chilled in Gilbert, C. S., Eppson, H. F., Bradley, W.B., and Beath, 0. A., ice water during the addition of the 83% sulfuric acid. Without Univ. Wyoming, Agr. Ezpt. Sta.. Bull. No. 277 (December chilling, results tend to be slightly lowvvhen this pigment is present. 1946). Holler, A. C., and Huch, R. V.. ANAL.CHEM.,21, 1385 (1949). In Table I are shown the standard deviation, the coefficient of Johnson, C . M., and Ulrich, A,, I b i d . , 22, 1526 (1950). variation, and the fiducial limits a t the 5% and 1% levels for samKlement, R., and Dmytruk, R., 2.ami. Chem., 128, 106 (1948). ples of Phalaris and wild oats which contain varying amounts of Kunin, Robert, and Myers, R. J., J . Am. Chem. Soc., 69, 2574 nitrate-nitrogen. The figures indicate that the proposed method (1947). Lugg, 6. W. H., Med. J . Malaya, 5, 140 (1950). is capable of yielding results of high precision. The final deterPiper, C . S., and Lewis, D. G., unpublished data. minations of the nitroxylenol were made with the aid of a Beckman RECEIVED for review July 31, 1952. Accepted January 5 , 1953. RIodel DU spectrophotometer. Table 1.
Precision of Nitrate Estimation
Colorimetric Determination of Potassium with Dipicrylamine ROGER FABER
AND
THEDFORD P. DIRKSE, Cahin College, Grand Rapids,Mich.
HE fact that the potassium ion forms an insoluble crystalline Tsubstance with dipicrylamine has been suggested as the basis for a gravimetric ( 2 , IO), conductometric (IO), and colorimetric (1, 3, 5, 6) method for the determination of potassium. For this laboratory the convenient and rapid method outlined by Amdur ( I ) seemed best. In this method the potassium is precipitated with a lithium dipicrylaminate reagent, the resulting mother liquor is drawn off and diluted, and its optical transmittance is determined. The larger the amount of potassium, the more dipicrylamine is precipitated. Hence, increasing amounts of potassium bring about an increase in the optical transmittance of the final solution. All the transmittance vaIues in the work re-
ported here were measured with a Coleman Cniversal spectrophotometer, using a wave length of 470 mp, It was soon discovered that there were limits with this method that had not been pointed out in previous reports. Specifically, zinc was a contaminant in solutions in this laboratory, and it was of importance to knoly the extent to which this introduced errors. The work of Kolthoff and Bendix (6) indicated that zinc might be an interfering ion, but the extent of the interference was not known. This paper is a description of some work carried out to determine the extent of zinc interference and also to evaluate other interferences. Any ion which brings about an appreciable precipitation of
809
V O L U M E 25, NO. 5, M A Y 1 9 5 3
c
i
0 0 2 M G
K
0 O3MG
K
According to Kinkel and Xaas ( I O ) , Sheintsis ( 8 ) ,and Kolthoff and Bendix ( 6 ) , ammonium ions must be removed, since they, too, form a precipitate x i t h the dipicrylamine. To determine the extent of this interference several series of solutions containing both potassium and ammonium ions were prepared. In each series the potassium ion concentration n a s held constant, but the ammonium ion concentration mas varied. These solutions a e r e then treated as they would be in the analysis for potassium. The results for the three series given on Figure l are typical. I t is evident that the presence of even small amounts of ammonium ion causes a considerable error in the potassium determination. The ammonium ions bring about the formation of a pale yellow precipitate which appears very quickly and in relatively large amounts. The potassium precipitate has a reddishorange color.
0 0 4 5 MG, K
0 . 4 5 MG. 40
-
K
0.40 MG. K
20
t 30 0.30 MG. K 20 MG. AMMONIUM S U L F A T E IO
Figure 1. Effect of .4mmonium Ions on Determination of Potassium
dipicrylamine, either as such or in the form of a dipicrylaminate, cannot be tolerated, since this will contribute to the change in the depth of color. I n addition to potassium, the ions of rubidium, cesium, aluminum, iron, chromium, nickel, cobalt, copper, bismuth, vanadium, titanium, thorium, mercury, thallium, and lead cause the precipitation of dipicrylamine from a solution of this reagent in strong alkalies. There is some disagreement as to whether those aside from rubidium, cesium, and potassium do so because they precipitate dipicrylamine as such (Y),or as the dipicrylaminate ( 4 9 ) .
I
I
I
I
0
HpSOq
0 0
HaPo,
I
SOLUTIONS
CH3COOH HCI
4 3
I
"
ZnSO,
i\
0
2
I
I
I 3
1
1
I
4 PH
5
6
Figure 2. Effect of Acidity on Optical Transmittance of Dipicrylamine Reagent
lot
Figure 3.
1 Effect of Alkalinity on Determination of Potassium
Khen solutions of dipicrylamine are acidified, the dipicrylamine is precipitated (4). This, of course, gives rise to errors. To determine more carefully the effect of pH on this particular method, several acid solutions Tvere prepared. The pH values were determined with a Beckmann Model H2 p H meter. The dipicrylamine reagent was added to these solutions ( 4 hich contained no potassium), and the mixture was allowed to stand for 2 hours. I t was then diluted, and the transmittance values xvere measured. An examination of these results (Figure 2) shows that the sulfate, chloride, acetate, and phosphate ions have no effect on the transmittance values. I n each case a pH of 3 or 3.5 is the lower limit for Accurate results. S o precipitation of the dipicrylamine takes place in acid solutions of pH 3 and above, hence the transmittance values of the solutions are unaffected. Several series of sodium hydroxide solutions were prepared, and in each series the potassium content was held constant. The pH values above 11 are calculated from the extent of dilutions and are approximate values only. These solutions were treated in the same oiay as were the acid solutions described in the above paragraph. The results, given on Figure 3, show that the upper p H limit for reproducible and accurate results is about 11. Above that, the increasing amounts of sodium ions probably cause the sodium dipicrylaminate to precipitate. This would cause an increase in the optical transmittance. Before this value increases, it first decreases, and the extent of this decrease varies with the potassium ion concentration. -1possible explanation is that the increasing concentration of sodium hydroxide exerts a dissolving effect on the potassium dipicrylaminate by causing an increase in the ionic strength of the solution. The presence of zinc ions produces a yellowish precipitate in addition to the regular reddish-orange precipitate of potassium
ANALYTICAL CHEMISTRY
810 dipicrylaminate. This is accompanied by erratic results for potassium. A series of solutions containing the sulfates of both potassium and zinc were prepared, and each solution was analyzed for potassium in the usual manner. The presence of zinc did increase the optical transmittance, but it appears that as the amount of potassium in the sample increases, more zinc ions can be tolerated without affecting the results for potassium. A safe and general rule in this instance is that only when the meight of zinc in a sample is greater than the weight of potassium need the zinc be removed or reduced in amount before proceeJing with the analysis for potassium. This rule has been followed in this laboratory and has given good results. Under such conditions no yellow precipitate is formed during the period the solutions stand before dilution. The zinc can be removed from the solution by precipitation with small amounts of a dilute sodium hydroxide solution. According to the thesis of Shapiro ( 7 ) , the yellow precipitate formed by the zinc ions would he dipicrylamine itself, and it is produced because the zinc salts hydrolyze, giving an acid solution. On this basis, the amount of yelloiv precipitate should be determined by the pH of the solution. However, it is a t once evident from Figure 2 that more than a pH factor is involved in the formation of this precipitate. If only pH were involved, then zinc sulfate solutions should have the same effect as acid solutions of the same pH. Honever, only a slight change in pH of the zinc sulfate solutions produces a large error in the potassium determination. This is in contrast to the effect of sulfuric acid solutions. When solutions containing only zinc sulfate TI ere treated with the lithium dipicrylamine reagent, a yellow-orange precipitate was formed, the amount of which increased n.ith increasing amounts of the dipicrylamine reagent. This precipitate was
but very slightly soluble in water. Its color was definitely different from that of plain dipicrylamine in the same solution. Kolthoff and Bendix (6) suggest that since the dipicrylamine reagent has an alkaline reaction, a cation such as zinc should yield a precipitate consisting of the hydrous oxide or some basic salt of zinc. There is definite evidence that this did occur. A close examination of the precipitate showed that there were two types of solid matter-the yellow or orange precipitate which may have been the zinc dipicrylaminate, and a smaller amount of a lighter colored, gelatinous material. This latter diswlved readily in dilute sulfuric acid and also in concentrated s3dium hydroxide solutions. This would indicate that this precipitate is a mixture of an oxide or hydroxide of zinc and some zinc dipicrylaminate. S o further study of this precipitate was made. ACKSOW LEDGMEKT
The xork reported here was carried out in connection xith a project sponsored by the Office of Saval Research. It is a pleasure to express thanks for this support. LITERATURE CITED
Amdur, E., ISD. ENG.CHmr., A N ~ LED., . 12, 731 (1940). Duval, C., Anal. Chim. Acta, 1, 105 (1947). Kielland, J., Ber., 71B,220 (1938). (4) Kielland, J., Ger. Patent 704,545, Feb. 27, 1941. ( 5 ) Kohn, W., 2. anal. Chem., 128, 1 (1947). (6) Kolthoff, I. M., and Bendix, G. H., IND.ENG.C H m r . , A N ~ L . ED.,11,94 (1939). (7) Shapiro, LI, Ya., Zacodakaya Lab., 7, 790 (1935). (8) Sheintsis, 0. G., Ihid., 4, 1047 (1935). (1) (2) (3)
(9) Ibid., 8, 1198 (1939). (10) Winkel, d.,and Maas,
H., Angew. Chem., 49, 827 (1936).
RECEIVED for review October 18, 1952. Accepted January 9, 1953.
Determination of Thiamine by the Thiochrome Reaction Application of Cyanogen Bromide i n Place of Potassium Ferricyanide MOTONORI FUJIWARA AND KIYOO XIATSUI Department of Hygiene, Kyoto L'niversity, h i o t o , Japan potassium ferricyanide and alkali reaction, which was T first proposed for the determination of thiamine in biological materials by Jansen (6) in 1936, is generally recognized as the HE
most useful reaction for converting thiamine to thiochrome, and is most commonly used for the determination of thiamine. I n 1949 Fujiwara (1) found that a blue fluorescent compound, "thiochrome," was produced on addition of alkali after thiamine was mixed with cyanogen bromide a t room temperature. This compound was proved to be thiochrome, melting point 227' C. (decomposes) by Matsukawa ( 6 ) , a staff investigator of the research laboratory attached to Takeda Pharmaceutical Industries, Ltl. The authors have studied the utilization of this reaction in the determination of thiamine in biological materials. in place of oxidation of thiamine with potassium ferricyanide and alkali. REAGENTS
Synthetic zeolite, prepared according to the method of Hennesqy ( 3 ) ; 25% potassium chloride in 0.1 S hydrochloric acid; isobutyl alcohol; 30% sodium hydroxide; anhydrous sodium sulfate; and cyanogen bromide solution. PROCEDURE
One and one half grams of activated zeolite are placed in an exchange tube which has an internal diameter of 7 mm. and a stopcock ( 2 ) . A finely pulverized sample, containing 3 to 5 micrograms of thiamine, is weighed and about 40 ml. of distilled water are
zddsd. This mixture is heated on a water bath at 80" C. for 1.3 minutes, being continuously stirred, after the pH is adjusted to 4.. After addition of about 3 ml. of 2% takadiastase solution and a few drops of toluene, the mixture is allowed to stand overnight in an incubator a t 38" C. Distilled water is added to make the total volume of liquid 50 ml. and the mixture is centrifuged a t high speed. A certain amount of clear supernatant solution, containing 1 to 2 micrograms of thiamine, is allowed to pass through the zeolite column a t the rate of about 1 ml. per minute after the pH has been adjusted to 4.5. The zeolite is washed with hot water, and then thiamine is eluted ivith 25 ml. of boiling potassium chloridehydrochloric acid solution. The eluate is made up to exactly 25 ml. in a graduated cylinder. A 5-ml. portion of the potassium chloride-hydrochloric acid eluate is transferred to a centrifuge tube, 3 ml. of cyanogen bromide solution are added and mixed, and 2 ml. of 30% sodium hydroxide are added and mixed. The thiochrome produced is extracted in 20 ml. of butyl alcohol in the customary manner. The blank is treated in the same way by omitting the cyanogen bromide. Fluorescence is measured in the Pfaltz & Bauer fluorophotometer according to the procedure of Hennessy and Cerecedo (4). CRITICAL STUDY OF STEPS IN PROCEDURE
Preparation of Cyanogen Bromide Solution. This reagent is prepared by adding an ice-cold 10% aqueous solution of potassium cyanide drop by drop to ice-cold saturated bromine water until it is decolorized. This reagent is stable for 3 hours a t room temperature (22" C.) as shown in Table I. Influence of Amount of Cyanogen Bromide Solution on Oxidation of Thiamine to Thiochrome. To decide the amount of
