456.
INDUSTRIAL AND ENGINEERING CHEMISTRY
The dropping electrode cell, ‘4, 11,~sa conical form with a maximum capacity of 5 mi. and is fitted with a ground joint, D, to stem C. This joint serves as the conductive connection with the surrounding calomel half-cell. Throuqh the center of the stem goes a tapered tube, B , ending in a capillary tip which serves to introduce the polarographically inert gas into the sdution. Side arm H is joined Lvith rubber tubing to the leveling bulb, K , which can be raised or lowered by the rack and pinion, L. A4t E and E , connections for the internal and external electrode are made. The calomel electrode is closed by a ground-in cup, F , and is sealed aqainst evaporation by a rubber band, G. The whole vessel is closed by a large ground-in cup, M , which has a protection valve, N. This cup is firmly attached to the stand a t l’, whereas the whole lower part with the leveling bulb c3n be moved up and down on the stand by a rack and pinion, R. The vessel is filled from a sma!l pipet with the solution t J be analyzed. By lowering the mercury with the levelin: bulb, K , so that the ground joint, D, is exposed to the solution but the tip of the gas-introducing capillary is still covered with mercury, the external electrode is ready to use as the anode. The graund joint has to be so shaped inside that no mercury ring is formed at the joint. The size of the capillary tip for the gas introduction must be small enough to keep mercury from leakinq down when bubbling of the gas through the capillary is stopped. The vessel is emptied by a siphon under s:iction, rinsed thorouzhly wit,li water, and dried with filter paper and a stream of dry air beforc the next solution is introduced. Because of the narrowness of the vessel a drawn-out capilla,,y,S , is used for the dropping electrode. This also must be wiped with filter paper after each analysis. This vessc~lW R P drveloped mainly for the purpose of trying out
Vol. 17, No. 7
microtitrations with the mercury dropping electrode as an endpoint indicator. For that purpose, the end of amicroburet, T , was introduced throuch the rubber stopper closing the large cup. This end of the buret was made from a 0.05-mm. bore platinum tubing and was inserted in the solution durinq titratiJns. The buret supplied 0.1 ml. of reagent on a scale 30 cm. long and the solution was displaced by mercury. The mercury was moved by 3, steel screw rotated in a Kovar member sealed to the qlass. Figure 3 shows. the arrangement used for titration work in combination with a compensation type of instrument. Preliminary experiments showed that volumes smaller than 1 ml. can be titrated in the veRsel with results within a few per cent of theoretical values. ACKNOWLEDGMENT
The vessels were skillfully constructed in 1940 by the late C. Kirwer. LITERATURE CITED
(1) Heyrovskg, “Polarographie.
Physikalisohe .Methoden der analytischen Chemie”, Teil 111, p. 438,Leipzig, Akademische S’erlaeseesellschaf t. 1939.
(3) Maassen, G., Z . angelc. Chem., 50, f Majer, V., Mikrochemie, 18, 74 (1935).
(4)
Determination OF Bismuth in Blood Serum or Plasma An improved Colorimetric Micromethod N. J. GIACOMINO1 Research Laboratories, Winthrop Chemical Company, Inc., Rensrelaer,
A n improved method is presented for colorimetric estimation 01 small quantities of bismuth in blood serum or plasma, it i s especially adaptable for determining from 10 to 100 micrograms per 100 cc. of serum. W h e n these amounts of bismuth are added to serum and 6’7’0,the error tending to b e plasma, the average recovery i s 99, less as the amount of bismuth increases. The principal improvements are: the use of ascorbic acid as a reducing and stabilizing reagent, the attainment of maximum light absorption, and the reduction to a minimum of the color intensity of the reagent blank.
*
S
INCE 1880 when Thresh ( 7 ) first published the iodobismuthite method for the estimation of bismuth, numerous attempts have been made to apply the procedure to the determination of bismuth in biological materials. The method of Leonard (3) may be regarded as the culmination of these attempts. Von Oettingen
(4)compiled a review of the several procedures and their modificntions published prior to 1930. More recently, new techniques ( 6 ) applicable to the determination of 5 micrograms or les3 in biological samples have been reported, great,ly facilitating research on the pharmacological behavior of this element. The method of Hubbard ( 2 ) is particularly accurate, but requires more involved manipulations than that of Sproull and Gettler ( 6 ) upon which the procedure presented in this paper is based. The iodobismuthite method depends in general upon the formation of a yellow complex in an acid solution, with the use of potassium iodide and a suitable reducing agent. However, the small quantities of bismuth ordinarily found in blood serum and cerebrospinal fluid yield colors too faint to be read in the usual laboratory photoelectric colorimeter. .I partial solution of this difficulty has been suggested by the‘ work of Haddock (I), who showed that the co1orc.d complex niny be extracted from an aque1
Present address, Yale University Scliool of Medicine, N e w Haven, Conn.
N. Y.
ous solution by a 3 to 1 mixture of amyl alcohol and ethyl acetate. A number of organic solvents and mixtures thereof have been tried in this laboratory, but Haddock’s mixture has been found as suitable as any for this purpose. The extraction of the iodobismuthite complex from aqueous solution into one fifth of the volume of organic solvent increases the sensitivity of the method fivefold. The use of a special absorption cell (Figure 1) further increases the sensitivity. , Because the reagent blank usually has an undesirably high color intensity, the possibility of using different reducing agents was given some attention. There are objections to using sulfurous acid as the sole reducing agent, since with potassium iodide it forms iodosulfinic acid, I(HSOz), which imparts a yellow color to the solution even in the absence of bismuth. The best results were obtained with a small amount of sulfurous acid supplemented by ascorbic acid as a second reducing agent. The ascorbic acid, furthermore, serves a dual purpose, since in preventing the slow liberation of iodine from potassium iodide i t stabilizes the extract for the time necessary to obtain the readings. The method reported herein, therefore, introduces several significant improvements in technique; the degree of accuracy and sensitivity which results is considerably greater than in the procedures hitherto reported. It is especially suited for determining from 1 to 10 micrograms of bismuth in 10 cc. of blood serum or plasma, but undoubtedly is applicable to determining very small amounts of bismuth in other biological materials, although no attempt has been made in the present work to demonstrate this point. REAGENTS REQUIRED
Standard bismuth solution, 0.2312 gram of bismuth nitrate pentahydrate dissolved in 1 to 10 nitric acid and diluted to I liter with distilled water.
ANALYTICAL EDITION
July, 1945
457
The usual straight line calibration curve was established by determining the light absorption of the iodobismuthite produced by known amounts of bismuth. The desired amounts of bismuth, taken from the bismuth standard, were treated with 5 cc. of 10 A' sulfuric acid. After dilution with 10 cc. of distilled water, the color development, extraction, and reading were accomplished by the method given below. PROCEDURE FOR ANALYZING BLOOD SERUM OR PLASMA
Figure
1.
Absorption Adapter
Cell
and
Length of absorption cell, 40.0 mm. along light axis. volumr, 6 cc. Outside dimensions o/adapter, 0.8 x 4.1 x 9 . 9 em.
Potassium iodide, 3%, 3 grams of C.P. potassium iodide (low in iodates) dissolved in 100 cc. of distilled water, (This reagent should be prepared daily.) .\scorbic acid, ly?,2 grams of good quality ascorbic acid dissolved in 200 cc. of distilled Xvater. (This reagent should be prepared daily.) Sodium sulfite, 0.75%, 0.75 gram of C.P. anhydrous sodium sulfite dissolved in 100 cc. of distilled water and 0.6 cc. of concentrated sulfuric acid added. (This reagent must be prepared daily.) Sulfuric acid, approximately 10 A-. .\my1 alcohol pure (Merck) and C.P. ethyl acetate, mixed in the proportion of 3 t o 1. Hydrogen peroxide, 30%, C.P. (Superoxol). Perchloric acid, 7070,and concentrated nitric acid, mixed in the proportion of 2 to 1 .
A 10-cc. sample of serum or plasma is ashed in a 100-cc. Kjeldahl flask according t o the procedure outlined by Sproull and Gettler with the modification that only 1.2 cc. of concentrated sulfuric acid are used to produce the proper hydrogen-lor1 concentration in the final solution. I t has been found necessary, also, to boil the digest twice with 10 cc. of distilled water until sulfur trioxide fumes appear a t the mouth of the flask, thus ensuring the complete removal of any excess oxidizing agents. Both the color development and the subsequent extraction can be carried out in a 50-cc. separatory funnel. The cold digest is transferred quantitatively to the 50-cc. separntory funnel Kith three 5-cc. portions of distilled ivater. After addition of 5 cc. of 1% ascorbic acid, the contents are mixed thoroughly and 2.5 cc. of the potassium iodide reagent are added with shaking. Finally, to complete the reduction of interfering substances, 1 cc. of the sulfite reagent is added. The solution is mixed thoroughly and allowed to stand for 10 minutes, then treated with 6 cc. of the amyl alcohol-ethyl acetate solvent and shaken vigorously for 2 minutes. After the contents of the funnel separate into 2 layers, the aqueous portion is tapped off and the extract run through a cotton filter-pad into the absorption cell. The reading is taken at once, using the extraction solvent to establish a zero point and deducting the value of the reagent blank from each of the other readings. The cell is rinsed with the solvent and nllowed to drain before each reading in order to remove any adhering solvent, from the previous determination.
Table Hisrnuth .\dried
Y
1
0.8 0 8 1.0 i n
r,
80
an
Standard Deviation
%
in
00
I00
I no f3 3 2
3.0 2 3 2.7 2 7
100 77 90 90
8.17
3.0 3.1 3.9 3.5 3.I
75 78 97 88 93
8.47
5
4.5 4.8 4.7 4.6
90 96 94 92
2.24
6
5,7 5.7 .5 . 8 5.2
95 9n 96 87
3.63
6.6 6.2 7.0 6,9
94 89
4.21
100
8
7.8 7.7 7.3 7,s
98 96 91 94
2.59
9
8.2 8.5 8.6 7.9
91 94 95 88
2.74
10
9.5 9.2 9.3 9.2
9: 92 93 92
1.22
DISCUSSION OF METHOD
JViegand, Lann, and Kalich (8) have demonstrated that the light absorption of the yellow iodobismuthite solution is maximal a t 460 mM wave length. Throughout this work, however, a filter with maximum transmission a t 470 mp wave length has proved entirely satisfactory in the Klett-Summerson colorimeter. The special absorption cell has a volume of only 6 cc., but causes the light to pass through 40 mm. of solution, thereby increasing the absorption readings about threefold over those obtained with the usual colorimeter tube. To hold the 40-mm. cell in place in the Klett-Summerson instrument (industrial model), a wooden adapter of suitable design was constructed (see Figure 1). Sproull and Gettler (6) have pointed out that the color intensity of potassium iodobismuthite is a function of the iodide concentration of the solution, and that the acidity of the solution and the amount of sulfite, within narrow limits, apparently have little effect upon light absorption, With 3y0 potassium iodide the iodide concentration is such that slight variations in measuring out this reagent do not appreciably impair the accuracy of the determination. Furthermore, the color intensity of the reagent blank was minimal with a 2 N hydrogen-ion concentration and with the sulfite reagent making,up 4% or less of the final volume. The amount of ascorbic acid may vary from 10 to 2070 of the final volume without affecting the color intensity.
I. Recovery of Bismuth
Ri-muth Recovered
I
98
Average recovery, 9 2 % Standard deviation of an individual value, *6y0
458
INDUSTRIAL A N D ENGINEERING CHEMISTRY DISCUSSION OF PROCEDURE
Since the color-developing reaction is very sensitive to oxidizing impurities, all glassware should be thoroughly cleaned with acid-dichromate cleaning solution or hot nitric acid, and rinsed several times with tap water and once with distilled water. As a final precautionary measure the glassware may be rinsed with a little of the 1% ascorbic acid solution, which will reduce any impurities present, such as dichromate from the cleaning solution. The most common interfering substance in this procedure is ferric iron. Sproull and Gettler have shown, however, that It must be prcscnt in excess of 20 mg. per 100 cc. to liberate iodine. The method can be carried out directly in the presence of considerable quantities of the alkali metals, magnesium, manganese, zinc, cobalt, nickel, chromium, and aluminum (6). As Wiegand, Lann, and Kalich have pointed out, using hypophosphorous acid instead of sulfite reagent, the sequence of reagent addition given above must be adhered to for satisfactory results. While it would be expected that ascorbic acid alone would exert sufficient reducing action, actual trials have proved that a little sulfite reagent must be present. Although the aqueous solution of potassium iodobismuthite is stable for scveral days if kept tightly stoppered, the extracted complex ha.; been found to remain stable for no longer than 30
Vol. 17, No. 7
minutes. The readings, therefore, must bc made without undue delay if accurate results are to be obtained. From 1 to 10 micrograms of added bismuth have been recovered from 10 to 20 cc. of normal dog serum and beef plasma with an average accuracy of 92 * 6%. These data, presented in Table I, demonstrate that the error ‘tends to become less as the amount of bismuth increases. If quantities of bismuth greater than 10 micrograms are present, it is necessary to use two or even three extractions with amyl alcohol-ethyl acetate; the extraction must be repeated until all the yellow complex has been removed from the aqueous solution LITERATURE CITED
(1) Haddock, Analyst, 59, 163-i (1934). (2) Hubbard, IND. Exo. CHEM., ANAL.ED.,1 1 , 343 (1939). (3) Leonard, J . Pharmacal., 28,81-7 (1926). (4) Oettingen, von, Physiol. Reu., 10,221-81 (1930). (5) Sandell, “Colorimetric Determination of Traces of Metals”. Vol. 111, p. 162,New York, Interscience Publishers, 1944. (6) Sproull and Gettler, IND.ENO. CHEM.,ANAL.ED., 13, 162-5 (1941). (7) Thresh, quoted by Scott in “Standard Methods of Chemical Analysis”, 4th ed., p. 79, New York, D.Van Nostrand Co., 1925. (8) Wiegand, Lann, and Kalich, IND.ENG.CHEM., ANAL.ED.,13, 912-15 (1941).
Separating and Detecting Cupric and Cadmium Ions in the Copper Subgroup of Group GERALD F. GRILLOT
AND
II
JERRY B. KELLEY
University of Kentucky, Lexington, Ky.
E
VANS, Garrett, and Quill (1) describe a method of separating cadmium from cupric ions by virtue of the fact that copper forms a complex tartrate which is soluble in an alkaline solution, whereas cadmium precipitates as cadmium hydroxide, (’lasses m elementary qualitative analysis at the University of Kentucky obtained unreliable results in using this test, as evidenced by the number of reports in which cadmium was missed. The students were cautioned to use more sodium hydroxide and a longer period of boiling, but little improvement in the detection of cadmium was noted. The authors have reasoned that it is. difficult to remove the ammonia completely by boiling; thus the cadmium remains in solution aa the ammonia complex ion. I t was decided to acidify with concentrated nitric acid the ammoniacal solution obtained in the separation of cupric and cadmium ions from bismuth. This was followed by evaporation to dryness in order to remove any ammonia or ammonium salts that were present. The residue was put into solution and was treated with sodium hydroxide and Rochelle salts solution, whereupon the soluble blue copper tartrate complex and a heavy white gelatinous precipitate of cadmium hydroxide formed. The authors recognize the fact that the cyanide method is as reliable as this method and much shorter. They, like many other instructors of qualitative analysis, object to the use of solutions of the poisonous alkali cyanides in large freshman classes of qualitative analysis. Therefore the goal in qualitative analysis seems to be the development of an alternative method which will bc as reliable and as rapid as the cyanide separation of copper nnd cadmium. This modification of the separation has given dependable rehults in a small class of pupils doing qualitative analysis, as well as in analysis carried out by the authors. Although this article dcscribes the method of separating cupric and cadmium ions
using the semimicrotechnique, it should be easily adaptable t o the macrotechnique. PROCEDURE
I t is suggested that the analysis as described by Evans, Garrett, arid Quill (f) be modified as follows: The decantate from the preci itation of bismuth hydroxide may contain Cu(SHa),++ and CJNHs),++. Copper is present if this decantate has a deep blue to purple color. If there is any doubt about the presence of copper, a small sample of the decantate can be acidified with dilute acetic acid and then 2 drops of potassium ferrocyanide can be added. A red precipitate of copper ferrocyanide confirms the presence of copper. If copper is absent, make the decantate just acid with dilute sulfuric acid and saturate the solution while cold with hydrogen sulfide gas. A yellowish precipitate of cadmium sulfide confirms the presence of cadmium. If copper is present, place the decantate in a casserole and acidify with concentrated nitric acid, then add 5 drops in excess. Evaporate this solution to dryness and heat until all ammonium salts are decomposed. Cool the casserole and add 1 to 2 drops of 6 N nitric acid and 1 ml. of water, Add 10 drops of 6 ili sodium hydroxide and l ml. of 0.5 iM potassium sodium tartrate (Rochelle salts) solution. Centrifuge. A white gelatinous precipitate of cadmium hydroxide confirms the presence of cadmium. The precipitate of cadmium hydroxide may appear to be blue. Centrifuge, decant, and wash twice with water. The precipitate should be white. A further confirmation can be carried out by just dissolving this white precipitate in dilute sulfuric acid and then saturating the cold solution with hydrogen sulfide gas. .4 yellow precipitate of cadmium sulfide confirms the presence of cadmium. LITERATURE CITED
(1) Evans, Garrett, and Quill, “Semimicro Qualitative Analysis", p. 54,Boston,Ginn & Co., 1942.
