Distillation Equipment Suitable for Centigram and Decigram Quantities CI,ARK W. G O U L D , JR.', G E O R G E HOLZMAN*, AND CARL NIEMANh California Institute of Technology, Pasadena, Calif. The simple and effective transfer of microliter volumes of distillate from condenser t o receiver through the use of capillary siphons is described. Stills embodying this principle have been used successfully for the fractional and molecular distillation of centigram and decigram quantities. $
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of the problems eiic.ouritei,edi r i the fractioiis1 dist,illatiuri
Lhis procedure, while useful itt tinies, saerificeu one or niow of the desirable features associated mitli fractional distillations conducted on a larger scale: convenient operation a t either atmospheric or reduced pressures, collection of a number of fractions without interruption of the distillation, and continuous indication o f the progress o f the distillation. ?
of centigram or decigram quantit,ies is the efficient transfer of distillate from condenser to receiver. Dropwise transfer vihich is suitable for stills of larger capacity (1, 2, 3 , b , 7 , 9, 10, 1aj is not feasible, as one drop of distillate may represent abut lo%, of a 200-microliter charge. I n :tddition it is ilesirahle that a microstill possess not only ion column holdup but also low holdup ~ to between still head and receiver, a feature n-liic.11K C J L I ~ appear ~ tho preclude the USCJ of stopcocks or trthw ground s u i , f : t c ~for (controlof the reflux ratio and the transfer of dislillwtc,. The technique used by LIortoti a i d ;\I:iliorlc.y j h ' ~ ; t i ~ d t)y Craig (4'1 for the collection of fractions in the distillation of decigram or smaller quantities is based upon the periodic removal of distillate from a single receiver with the aid of a capillary piyjet.
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1 Present addrebs, Central Research Lahoratorj-. Genera1 Aniline and Yilm Corp., Easton, Pa. Present address, Department of Chprnistry. lIa,sarhusetts Iuhtitute of 'I'echnolopy, Camhridpc
'
bL Figure 2.
Semimicrostill
Therrnocouple well. b. E n d of jacket heating coil. Condenser. d. Replaceable receiver cups, 3 X 20 mm., o.d. c . Receiver holder, 6 mm. o.d., t o hold four receiver cups. f . Inner joint c u t t o give adequate clearance for receiver holder. g. Capillary tube, 0.2-mm. bore. h . To uavuum sy?tem
a.
c.
Detail of Condenser Receiver System
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In this paper sevrral stills are described which operate effectively a t both atmospheric and reduced pressures and which permit the efficient collr.ctioq of microliter quantities of distillate up to a maximum of four fractions without interruption of the distillation. Provision is also made for the measurement of the temperature a t the still hrLad with reflux rates as low as 5 ~ 1 . per minute. Although these stills possess low holdup, control of the reflux ratio is limit,ed t o that which is possible by ca.reful control of the temperature o f tlir t) i t h surrounding the boiler, and of the condenser housiiig,
m
Figure 1. Microstill
BiIcttosrILL
Condenser inlet. 6. Condenser outlet. c. To vacuum system. d . Cold finger condenser. e. Collecting t i p for condensate. f. Capillary tubes, 0.2-mm. bore. g . Receivers. capacity 45 pl. h. Ends of condenserhousing heater roils, G t o 7 turns spaced 4 to 5 mm. a p a r t of No. 28 ChromelA wire. 1. Thermocouple well. j . Column jacket evacuated to 10-8 mm. and eilvered t o leave clear strips 2 mm. wide in 'front a n d back. k. Column tube, 2 5 mm. i.d. X 150 mm. 1. Packing, 3 t o 4 turns per cm. of No. 30 Chromel wire. m. Boiler. capacity s u 5 c i e n t for 100- t o 300-pl. charge. _n. Column heater coil, 8 t o 9 t u r n s spaced 12 to 13 mm. a p a r t of No. 28 Chromel-A wire
The microstill is illustrated in Figure 1. The condenser-receiver system is so constructed that the condensate accumulates at the collecting tip, fills the 0.2-mm. bore capillary tube which is in incipient contact with this tip, and thus passes into the receiver. At the recommended distillation rate of 5 to 20 pl. per minute the surface of the conden3er will appear dry, yet distillate -n-ill flow steadily into the receiver. At higher distillation rates a drop of distillate may form on or near the collerting tip, flow down the
a.
361
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ANALYTICAL CHEMISTRY
362 outside of the capillary tube, and thus escape collection in the receiver. The dimension of the capillary tube must be such as to avoid rupture of the liquid column during the course of a distillation. For liquids of high density and low surface tension-e.g., carbon tetrachloride-the maximum length of a capillary tube 0.2 mm. in inside diameter is about 18 mm. The receivers, each 2.5 X 15 nim., are mounted upon a supporting rod sealed to a female 14/20 standard taper, so that as the joint is rotated, the center of the top of each receiver will pass under one side of the collecting tip. The tops of the receivers are constricted so as to leave a hole about 1 mm. in diameter; this constriction serves to center the capillary tube in the receiver and to minimize loss of distillate by diffusion. The amount of distillate in a receiver may be estimated visually to n-ithin 10 to 20% or determined more accurately with the aid of a telescope equipped with a filar eyepiece. Fractions are removed from the receivers with weighed capillary pipets.
T S 10/30
b
v Figure 4. I f o l e c u l a r Still
Condenser inlet. b. Condenser outlet. r . To vacuum system. d. Still housing. e . Cold finger condenser. f . Flanee for collecting condensate. 8 . Capillary tubes, 0.2-mm. ,bore. i . Receivers, capacity 45 pl. 2. Boiler, capacity 200 rl a.
when tested with carbon tetrachloride-benzene mixtures (8). The performance of the still in several typical distillations may be judged by the data given in Figure 5 . SE3IIMICROSTILL
L w e i 1 for 50wott '-Thermometer Chromolox cortridq
Figure 3 .
wells'
Cavity for both liquid
1
Heating Block
The fabrication of the condenser-receiver system illustrated in Figure 1 is difficult, though a t least one manufacturer (Ace Glass Co., Vineland, S. J.) has made a number of satisfactory stills of this construction (11). The straight condenser of the semimicrostill (Figure 2) is considerably easier to construct and in principle would appear to be reasonably satisfactory for use in the microstill. The column, 2.5 mm. in inside diameter X 150 mm. with a wall thickness of 0.2 mm., contains a tightly fitting helix of 3 t o 4 turns per em. of S o . 30 Chrome1 wire. The column is surrounded by a jacket 15 mm. in diameter, evacuated to 10-5 mm., and silvered so as t o leave tn-o transparent strips, 2 mm. wide, so aligned rn to permit observation of the column during a distillation. The heating coils on the column jacket are not ordinarily used during a distillation but serve to remove less volatile substances from the column after a distillation has been completed. At ordinary distillation rates and xhen the condenser-housing coils are not in operation the 5- to 10-mm. section between the jacket ring seal and the side arm functions as an air condenser, thereby permitting conditions approximating total reflux. When the condenserhousing coils are placed in operation, the temperature of the condenser housing and adjacent regions may be raised to the point where a fraction of the vapors will pass into the side arm and be collected on the condenser. The temperature at the still head may be measured with the aid of a single-junction iron-constantan (4 mil) thermocouple inserted into the thermocouple well, 0.5-mni. bore and 0.1-mm. wall thickness, which extends 5 to 10 mm. below the top of the silvering of the column jacket. At reflux rates of more than 5 111. per minute accurate temperature measurements may be made. Distillation rates of 5 to 20 pl. per minute can be maintained with a quiet distilland surface and for this reason no boiling aid is required. Accessories needed for the operation of the microstill include a vacuum line, a potentiometer, and a heating block. The block (Figure 3) is used as an oil bath for the microstill or horizontally as a n air bath for the molecular still (Figure 4). The temperature of the block is controlled with the aid of a variable autotransformer. The microstill was found to have an efficiency equivalent to ca. 9 theoretical plates with a column holdup of 20 to 30 p1.
The semimicrostill (Figure 2) is simply a larger version of the miorostill, except for the modifications in the receiver housing. The column is 300 mm. in length and 5.5 nim. in inside diameter, and is made of Pyre.; thin-walled tubing. Without any packin'g the column showed about the same efficiency as the microstill. In the mi micros till, because of the need for greater flexibility, the bo r flask is attached to the column with a standard taper. A seco 1 and desirable modification in this still is the use of replacea :receiver cups (capacity about 150 PI.) which obviated 100
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40 60 80 Volume Percent Distilled Figure 5 , Performance of ,Microstill
100
0 . 1:1 mixture of 2-chlorovinyldichloroarsine and benzyl chloride; vol. of distillate, 160 pl.; pressure, 10 t o 25 mm.; b a t h temp., 96.5-12Z0 C ; time, 160 min. 0. 1: 2 mixture of 2-ohlorovinyldichloroarsine and bis(2-chloroethyl) sulfide; vol. of distillate, 283 id.; pressure, 1.5 t o 10 mm.; b a t h temp., 92-127O C . ; time, 210 min. 0 . 2 : l mixture of 2-chlorovinyldichloroarsine and crude bis(2-chloroethyl) sulfide; vol. of distillate, 293 pi.; pressure, 2 t o 10 mm.; b a t h temp., 115-125' C: time 240 min. X . 1.8:l'mixt;re of carbon tetrachloride and benzene; vol. of distillate, 213 p l pressure, 745 m m ' b a t h temp., 92-98' C:; time, 200 min. Com'position of fractions determined by specific gravity measurements
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V O L U M E 20, NO. 4, A P R I L 1 9 4 8 the use of a number of receiver assemblies in case more than four fract,ions were desired. %lOLECULARSTILL
The receiver of the molecular still (Figure 4) is identical with t h a t of the microstill. The distillate is collected upon the flanged section of the straight, inclined cold-finger condenser and transferred to the receivers with the aid of capillary tubes 0.2 mm. in inside diameter. I n operation, the boiler, containing a 50 to 150-111. charge, is inserted into the cavity of the heating block clamped with its long axis horizontal. At the pressure obtainable with a diffusion pump, or v i t h a good mechanical pump, high boiling oils-e.g., dibutyl phthalat,e-could be distilled a t a rat,e of 20 pl. per minute at temperatures belmv 50' C. when ice water was passed through ?he condenser. LITERATURE CITED
(1) Baker, R. H.. Barlieribus, C., and Roswell, C. A , ISD. ESG. C H E M . . .4X.\L.
En., 12,408 (1940).
363 ( 2 ) Bower, J. R., Jr., and Cooke, L.
M,, Ibid., 15, 290 (1943). (3) Cheronis, N. D., and Levin, N..J . Chem. Education, 22, 86 (1945). (4) Craig, L., IND.ENG.CHEY.,ANAL.E D . ,9, 441 (1937). (5) Lecky, H. S., and Ewell, R. H., Ibid., 12, 544 (1940). (6) Morton, A. A., and Mahoney, J. F., Ibid.,l3,494 (1941). (7) Naragon, E. A., and Lewis, C. J.,Ibid., 18,448 (1946). (8) Rosanoff, M. A., and Easley, C. IY., J . Am. Chem. SOC., 31, 953 (1909). (9) Selker, M. L., Burk, R. E., and Lankelma, H. P., IND. ENG. CHEW,AXAL.ED., 12, 352 (1940). (10) Shrader, S. A , , and Ritzer, J. E., Ibid., 11, 54 (1939). (11) U. S. Dept. Commerce, OTS,PB 5951 (1946). (12) Weston, P. E . , IND.ENG.CHEM., AXAL.E D . ,5, 179 (1933). RECEIVED October 27, 1947. Contribution 1155 from the Gates and Crellin Laboratories of Chemistry, California Institute of Technology, Pasadena, Calif. Based upon work done for Office of Scientific Research and Development under Contract OEhIsr-325 with the California Institute of Teehnology.
Microdetermination of Bismuth in Biological Material A n Improved Photometric Dithizone Method DOS.ILD 31. HUBBARD Ketterirag Laboratory of .4pplied Physiology, College of .I.ledicine, Unicersity of Cincinnati, Cincinnati, Ohio . i n improved photometric dithizone method has been developed for the determination of bismuth in biological material, applicable to 100 ml. or less of urine and 20 grams or less of blood. Bismuth is originally isolated from the highly diluted digested material, using a solution of dithizone in chloroform (60 nig. per liter), lead is separated with a buffer solution of pH 3.4, and bismuth is finally determined
A
N IAIPKOVED photometric dithizone method for the determination of bismuth in biological material has been developed recently and applied in this laboratory. Following preparation of samples by the regular n-et-ashing procedure ( 5 ) ,bismuth need not be isolated from mixtures of extraneous salts as the sulfide ( 4 ) , but can be extracted directly and qua,ntitatively as bismuth dithizonate. Separation from the lead dithizonate extracted simultaneously is effected a t pH 3.4, as indicated by Bambach and Burkey ( I ) , the final estimation of bismuth being made a t high pH, as described by Snyder (6). The sensitivity of the procedure is equivalent to that of the sulfide separation (4). REAGENTS AND APPARATUS
Ordinary high-grade chemicals m3y be used throughout the method. Eastnian dithizone may be used without further purification. The chloroform used may be a freshly obtained product after redistillation from Pyrex or used chloroform reclaimed by a procedure previously described ( 1 ) . All Squibb-type (PJ-rex) separatory funnels used must be cleaned meticulously with hot 50uc by volume nitric acid and distilled water to ensure removal of any bismuth or lead present as surface contaminntion from previous use ( 4 ) . The same applies t o the matched pairs of cells used for density measurements. Any standard spectrophotometer ma>- be used for nieasurenients of density. Instruments must be capable of isolating a narrow band centered a t 505 mM. PROCEDURE
Preparation of Samples. Samples of biological material, 100 nil. or less of urine, or 20 grams or less of blood or tissue, are prepared for analysis by a n-et-ashing method ( 5 ) . The digestion is made in a distilling flask, 1 liter in volume, provided with three necks possessed of interchangeable ground-glass connections, into
spectrophotometrically. The accurac? is within *0.1 microgram of bismuth for the range 0 to 10 micrograms and *0.5 microgram for the range 0 to 50 micrograms. Only one strength of dithizone solution is used throughout the complete procedure. Although used specifically for urine and blood, the method can be applied to other biological material. Bone samples may require special treatment.
n-hich are fitted the distilling head and txvo separatory funnels. This method of digestion involves the addition of 20 ml. of concentrated sulfuric acid (specific gravity 1.84), 5 ml. of perchloric acid (70 to 727,), and 20 ml. or more of nitric acid (specific gravity 1.42). The di ested sample is allov-ed to cool, 50 ml. of distilled water are afded, and the solution is transferred quantitatively to a 400-ml. Pyrex beaker. The beaker and contents are placed in a n ice water bath, 15 ml. of 407, w j v ammonium citrate solution and 50 ml. of 20% TV/V sodium sulfite solution are added; then, with stirring, 100 ml. of ammonium hydroxide (specific gravity 0.9) are also added. Extraction 1. The prepared sample is transferred quantitatively to a 500-ml. Squibb-type separatory funnel. After the addition of 5 ml. of 107, w/v potassium cyanide solution, the mixture is diluted to 400 ml. with distilled water and mixed thoroughly. Bismuth and lead are extracted as the dithizonates by successive additions of 10-ml. portions of dithizone in chloroform (60 nig. per liter) until the last portion shows no color change. For amounts of bismuth not exceeding 10 micrograms, three portions of dithizone solution suffice. If the amount of bismuth exceeds 10 micrograms but is not over 50 micrograms, four portions of dit,hizone solution are necessary. Each additional 10-ml. portion is roughly equivalent to 50 micrograms of bismuth. The combined extract is collected in a 125-m1., graduated, Squibbtype separatory funnel. Depending upon the amount of bismuth present, either the whole or an aliquot of the total previous estract, containing not more than 50 micrograms of bismuth, is taken for Extraction 2, and washed wit,h 50 nil. of distilled mater. after separation of the tn-o phases, t,he chloroform phase is transferred to a clean separatory funnel and the aqueous phase is shaken with 5 ml. of dithizone in Chloroform (60 mg. per liter); the latter is added to the chloroform phase but the \\-ash water is discarded. Extraction 2 'Removal of Lead.) The chloroform phase containing bismuth and lead dithizonates plus excess dithizone is shaken for 30 seconds with 50 ml. of buffer solution, pH 3.4 (I), and the chloroform phase is transferred to a clean separatory
