JULY 15, 1935
ANALYTICAL EDITION
This apparatus will control the pressure within I mm. and its over-all range i s l i m i t e d only by the depth of the U-tube, up to 760 mm. In order to set the regulator for the r e q u i r e d pressure, the p o s i t i o n of the U-tube with respect to knife-edge N is first adjusted by sliding beam M through the sleeve by means of which it is attached to the knifeedge N until a p p r o x i mately the required pressure is obtained in the system as measured by an independent manometer. When the required presFIG~JRE 111. PHOTOGRAPH OF s u r e i s o n t h e system, APPARATUS beam should be in an aPProximatelY horizontal position. Further adjustment is obtained by means of G and the final regulation may be controlled by means of D.
285
The operation of the assembly is as follows: As the air is exhausted from the system, the mercury in the Utube is drawn up in the left leg only a fraction of the distance which it drops in the right leg; hence a maximum weight of mercury crosses the center of gravity and causes beam M to swing on the knife-edge in such a way as to raise the rubber valve seat, thus allowing air to bleed in through B. As the vacuum decreases the beam swings back tending to close B. Actual1 , in operation, the rubber face on the plate attached to lever C L a t s an infinitesimal distance away from B, never actually shutting it off. This results in very smooth operation, as evidenced by the fact that the oscillation in mercury meniscus of the manometer approaches the limits of ordinary vision. The size of the bore in capillary B should be chosen rather larger for low vacuums than for higher vacuums. A little experience will suggest the size of aperture to use in order to obtain maximum sensitivity.
Literature Cited (1) Jacobs, G.W.,IND.ENG. CHBM.,Anal. Ed., 7,70 (1936). (2) Palkin, S.,and Nelson, 0. A., Ibid., 6, 386 (1934). RECEIVED March 4, 1935.
Determination of Lead Removal of Bismuth Interference in the Dithizone Method C. E. WILLOUGHBY, E. S. WILKINS, JR., AND E. 0. KRAEMER Tumor Clinic of Jefferson Hospital, Philadelphia, Pa.
I
N ANALYZING biological materials such as blood, excreta, tissues, etc., for their lead content by a previously described dithieone method ( I ) , bismuth, if present, seriously interferes, inasmuch as it reacts with the dithieone reagent under the same conditions as specified for lead. Bismuth may often be present in specimens of this kind as a result of previous medication. In order t o extend the applicability of the dithizone method for lead to materials also containing bismuth, the procedure described below was developed and incorpoirated in the original method. Bismuth is separated from the lead before its final estimation by extracting a nitric acid solution of the two metals, which has been adjusted t o pH 2,0, with a chloroform solution of dithieone. Whereas lead does not react and remains in the aqueous phase, bismuth reacts with the dithieone reagent and forms a chloroformsoluble organic complex. B y means of this additional step, amounts of lead ranging from 0.002 t o 0.200 mg. have been satisfactorily recovered from samples of lead and bismuth nitrates containing 0.500 mg. of bismuth. Larger quantities of both lead and bismuth could, in all probability, be handled with equally good lead recoveries.
Procedure GLA.SSWARE AND REAQENTS.These are the same as previously specified ( I ) , with two exceptions. The concentration of dithizone solutioh 1may be increased to facilitate the separation of appreciable quantities of bismuth. The solution should not be so concentrated, however, as to mask the color changes during extractions. With 0.50 mg. of bismuth, for example, a solution containing 0.20 gram of dithizone per liter is suitable. Secondly, another indicator, acid mcresol purple, standardized 0.04 per cent solution, is required. PR~PARATION OF SAMPLE AND SIMULTANEOUS EXTRACTION OF LEADAND BISMUTH.The destruction of organic matter, the removal of lead and bismuth simultaneously from the digest with dithizone solution 1, and the conversion of the lead and bismuth from dithizone complexes to the corresponding nitrates is carried out exactly as described in the original method ( I ) . SEPARATION OF BISMUTH.Two drops of acid mcresol purple are added to the nitric acid solution, which is then adjusted to pH 2.0 by the addition of 5 per cent ammonium hydroxide. At this
point the solution should have a volume of 25 to 35 ml. The solution is extracted with 25 ml. of dithizone solution 1. Inasmuch as the reaction between an appreciable amount of bismuth and dithizone is rapid, the separatory funnel need not be shaken vigorously for more than 2 minutes. After complete settling, the chloroform phase is removed and discarded, 0.2 to 0.4 ml. of the chloroform solution being left in the separatory funnel, to guard against any loss of the aqueous phase. The aqueous solution is reextracted twice more with 5-ml. portions of the dithizone solution. In each of these extractions the separatory funnel is shaken vigorously for 5 minutes to insure the removal of the last traces of bismuth. With 0.5 mg. of bismuth, the first portion (25 ml.) of dithizone solution is changed to a deep wine-red color by the reaction, the second portion (5 ml.) shows little, if any, color change, and the third portion (5 ml.) remains entirely unchanged in color; this indicates that the removal of bismuth is complete. Whether or not a color chan e has occurred is more easily observable during the shaking of t f e separatory funnel than after the subsidence of the chloroform phase. Larger amounts of bismuth would be removed in like manner by extracting with successive increments of the dithizone reagent until a newly added portion undergoes no change in color. Following the bismuth removal, the aqueous phase is
TABLEI. DETERMINATION OF LEADIN SOLUTIONS CONTAININQ 0.5 Ma. OF BISMUTH AS THE NITRATE (In these experiments the lead, as lead nitrate, was added by another chemist in amounta unknown t o the analyats.) Total Lead Lead Found Added Lead Lead Added Found in Blank Recovered Error MQ. MQ. MQ. Mg. Mg. 0,002 0.005 0.008 0.023 0.060 0,100 0.110 0.150 0.160 0.170
0.180 0.190 0.190 0.200 0.200
0.004 0.008
0.010 0.024 0.062 0.103 0.113 0.152 0.162 0.171 0.181 0.193 0.192 0.201 0.203
0.002 0.004 0.002 0.002 0.002 0,002 0.004 0,002 0.003 0.002 0.002 0.003 0.003 0.002 0.003
0.002 0.004 0.008
0.000 -0.001
0.022 0.060 0.101 0,109 0.150 0.169 0.169 0.179 0.190 0.189 0.199 0.200
-0.001 0.000 +0.001 -0.001 0.000 -0.001 -0.001 -0.001 0.000
0.000
-0.001 -0.001 0.000
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INDUSTRIAL AND ENGINEERING CHEMISTRY
washed with small portions of chloroform to remove the dithizone completely. A chloroform trap must be maintained continuously. LEADESTIMATION BY TITRIMETRIC EXTRACTION. The aqueous lead nitrate solution is now ready t o be prepared for the titrimetric extraction in which the lead content is determined. Two to three drops of phenol red, standardized 0.02 per cent indicator solution, and 2 ml. of 10 per cent potassium cyanide solution are added and the pH of the resulting solution is adjusted to 7.5 with 5 per cent nitric acid. (The residual mcresol purple does not interfere.) The solution is then extracted with standardized dithinone solution 2 as described previously (1). As in the original method, combined reagent blanks, consisting of the same amounts of all the reagents used in the analysis, are carried along simultaneously with the sample and in identical apparatus in order to correct for lead added from this source.
VOL. 7, NO. 4
Experimental Results The data in Table I are representative of the results obtained. It is evident that the separation of bismuth does not affect the accuracy with which lead is recovered by this method.
Literature Cited (1) Wilkins, E. S., Jr., Willoughby, C. E., Kraemer, E. O., and Smith, F.L., 2nd, IND.ENG.CHEM.,Anal. Ed., 7, 33 (1935). RECEIVED June 6, 1935. This work was supported by The Elizabeth Storck Kraemer Memorial Fund created by Pierre S. and Lammot du Pont.
Permanent Aqueous Microscopic Mounts H. R. SMITH, National Canners Association, Washington, D. C.
R
ESEARCH and instruction in many branches of the biological sciences call for microscopic examination of specimens of materials, many of which have to be in aqueous mountings. The lack of a quick and easy means for preparing permanent mounts of such specimens has made it impractical to assemble sets of standard slides for comparison, exhibition, and instruction, and has also delayed work in the field of microscopic study of slow reactions in situ. The need has been for a suitable sealing material to prevent evaporation. The desired preparation must adhere tenaciously to smooth glass surfaces, flow freely into place, not dissolve or diffuse in water, not dry out or crack on long standing, and be firm enough to hold the mount in place. Many products were tried, such as Canada balsam, shellac, rubber, cement, beeswax, Halowax, asphaltum, glycerolgelatin compound, etc., but each one failed to have all the necessary properties. Finally the suggestion of Parker (1) led to the right combination. Wool fat (lanolin) is a sticky animal wax, but being rather soft it has to be hardened somewhat. The sealing material recommended is made by heating anhydrous wool fat (adeps lanae, U. S. P.) with not more than 20 per cent of rosin, until the constituents are blended. (A few minutes over a Bunsen flame are sufficient.) This mixture is firm a t ordinary temperatures but on being heated it becomes liquid. It contains no volatile solvent and quickly congeals to hold the mount in position. Any microscopic mount may be preserved by sealing the edge of the cover slip with the melted wax. The space under the slip should be filled with liquid, although a few air bubbles do not interfere with the permanence of the seal. The slide and cover slip should be dry a t the points where the wax is to be applied. The melted wax may be applied with a thin glass rod or small brush, care being taken to have the wax come on top of the slip all the way around. Unless the slide is intended for the study of progressive reactions of reagents, growth of microorganisms, etc., the water used in their preparation should contain about 0.2 per cent by volume of 40 per cent formaldehyde solution. A number of uses for such permanent mounts have come to the author’s attention. A series of slides showing successive stages of a biological process may be prepared for classroom instruction, and once prepared they are available a t all times. Preparation of such slides may be made part of students’ laboratory work, and excellence of technic encouraged by making the best slides a part of the permanent collection. Special or abnormal tissues may be kept for reference and further study. Observations by one investigator concerning
a particular tissue may be reviewed by another investigator in some distant laboratory more understandingly if the comments of both are directed toward the same tissue on a permanent mount which is sent from one to the other. Research workers may be able to follow with the microscope the slow reaction of a reagent on a biological tissue, diffusion through the walls of unbroken cells, the growth and multiplication of microorganisms, or the growth of crystals. The same microscopic structure may be examined a t intervals over a period of days, weeks, or months. An application of the use of permanent slides for instruction purposes was developed by Wildman (a) in connection with the Howard mold-count method for tomato products. He found it necessary to devise two additional features: (1) the thickening of the sample so that the filaments would not change position on the slide; and ( 2 ) the designation of specific fields on the slide for examination. The sample was thickened by stirriqg into the hot sample about one-third its volume of hot 3 per cent agar solution. The particular fields to be examined were designated by a pattern made by punching in thin transparent colored Cellophane 25 holes, each exactly the diameter of the microscopic field. This pattern was first mounted in balsam on the slide beneath a cover slip and the prepared sample was spread on top beneath a second cover slip and sealed. Wildman used balsam for sealing the mount, but this is difficult to apply, slow to harden, and the seal is not always permanent. The wax described above has been used with entire satisfaction in the preparation of a number of such slides for the Howard mold-count method. An additional operation has been included in the examination of these slides which increases their usefulness for instruction purposes : A speciaI reporting sheet having 25 circles each about 1 inch in diameter, arranged in the same order as the holes in the pattern, is used to record the observations of each observer. The analyst sketches in each circle a representation of the relative position and size of each piece of mold he finds in the corresponding microscopic field. After each analyst has recorded his observations on a separate reporting sheet, the results may be reviewed and further examination made of particular filaments.
Literature Cited (1) Parker, B. W., Science, 80, 456 (1934). (2) Wildman, J. D., Ibid., 77, 263 (1933).
RECEIVED June 26, 1935.
