ANALYTICAL CHEMISTRY
1492 tion or by intravenous injection, :inti extracted thc 01’g:niis of the animal with alcohol. IVhen this alcoholic extract, whicli contained traces of chloroform, was mixed with a 10% aqueous ~ o l u tion of sodium hydroside and 2 ml. of pJ.ridiiie : ~ n d t~oilcd,n bright blue-red coloration dcvelol)ctl inimcdiati~l~~. Cole ( 2 ) estended this color reaction to a colorinictric dc~tcriiiinationof chloroform. He could detect 0.00017, of chloroforni in an q u r OUR solution or in an extract of animal tissues. A new color reaction, suggested h!. Fujiwarn’s discovery, has been elaborated by the author, and is dcscribetl here. IVhen :I compound that contains methyl ketone or methyl carbinol groups is treated with alkali hypochlorite or hypobromite and the whole is heated with the addition of pyridine, a carmine red or pink color immediately develops. In this reaction pyridinc is not the fiole reagent; pJ.iidine, quinoline, and their homologs are also available. Hypoioditc, however, is not applicable i n lieu of hvpochlorite or hypohwrnitc. For conveiiicncc the reaction with hypochlorite is called the “chloroform rc:iction” (C) and that with hypohromite the “bromoform reaction” (13). The sign means a positive color reaction and the sign ” - ” dcnotes no caolor reaction. Three different cases were csamined. Compounds Showing Positive Iodoform Reactions. Acetone, ethanol, acetaldehyde, isopropyl alcohol, acetophenone, p-hydroxyacetophenone, aeetoveratrone, acetopiperonc, and ~V-acetyl-a,p-diphenglindol. Both bromoform and chloroform react’ions were positive in all cases. Compounds Containing Neither Methyl Ketone nor Methyl Carbinol Groups, but Showing Positive Iodoform Reactions. Styrasinolic acid (6) (B+, C+), acetyl styraxinic acid (6) (B+ C - ) &,a-dibenzoylethane (7) (B+, C+), cholesterol ( 1 ) (b+, dibenzalacctone ( 1 ) ( B + , C f ) , &-phenyl-[& methyl, phen)-l]-ethyle~ieglycol( 1 ) (B+, e+),citric acid ( 7 )
”+”
6-),
(B-, C-).
Compounds Containing Neither Methyl Ketone nor Methyl Carbinol Groups and Showing No Iodoform Reactions Consequently. Thirty-four compounds of different constitutions
were chosen a t random. Of these, only carbazole showed a positive bromoform reaction but no chloroform reaction. Owing to the fewer exceptional reactions observed, thc chloroform and bromoform reactions seem to esccl the iodoform reaction. EXPERLMENTAL
Preparation of Hypochlorite Solution. Bleaching powdor ( 4 grams) wm mixed with water (25 ml.), and sodium carbonate solution (6 grams of sodium carbonst,e with water of crystallization and 15 ml. of water) was added and well stirred. The resulting calcium carbonate was filtered off. The slightly yellow solution thus obtained was applied for the chloroform reaction. Preparation of Hypobromite Solution. Bromine (2 grams) was dissolved in 50 ml. of 0.6 hr potassium hydroxide solution. The resulting solution was available for the bromoform reaction. Manipulation of Analysis. A small amount of t’he compound which contains the methyl ket.one group (one drop, if liquid) was taken in a t,est tube, 1 drop of hypochlorite (chloroform reaction) o r hypobromite (bromoform reaction) solution and 2 drops o f pyridine were added, and the whole was warmed over a tree fame. A carmine red or pink color promptly m:icle its aprmr:111ce. ACKNOWLEDGMENT
The author is indebted to Sin’iti Eiawai, a t whose suggestion this work was undert’aken and who supplied many valuable spwimens. LITERATURE CITED
(1) .kclachi. Jiro, unpublished data. ( 2 ) Cole, W. H., J . Bid. Chem., 71, 173 (19%). (3) Fujiwara, K., Sitzber. Naturw. Gea. Restock., 6, 33 (1916). ( 4 ) Kamlet, Jonas, 1x11. ENG.CHEX, A w \ r . . Eo.. 16, 3132 (1944). (5) Kawai, S., e t al., Ber., 72, 369 (1939). ( G ) Ibid., p. 1146. (7) JIeyer, Hans, “Analyse uiid Iionstitut.ionscrmittluiig organischer Verbindungen,” Gth Aufl., pp. 305. 306. Berlin, Julius Springer, 1938. RECEIVED December 1, 1950.
Determination of Nitrogen by Micro=Dumas Procedure EL3IEH F. SHELBERG iibbott Laboratories, .\*orth Chicago, 111.
’M-ERAL modifications of the original micro-Dumas inrthotl have been described recently (1-4, 7’). The usual Dumas method ordinarily furnishes erratic result,sunless the sample burning and carbon dioxide flow are carefully controlled. A proceclure currently in use at Abbott Laborat,ories obviates the necessitj. for slow sample burning and slow gas flo~r-. The analysis nisi. tw completed in approsimately 10 minutes (not including ~r-eighinp time) and has provided results \Tithin 0.1% of theory on hundreds of analyses by scveral analysts. This fast procedure involvcs the use of two combustion tubes, and is particularly adaptable to routine analyses in rapid succession, as it is not necessary to cool the tube that is heated in the furnace between determinations. APPARATUS
T h e apparatus for the rapid Dumas procedure is assernhltd illustrated in Figure 1.
:LP
Carbon Dioxide Generators. Two standard dry ice generators or chemical generators, A and A’, include stopcocks 1 :1nd 5 , respectively. Removable Combustion Tube. A standard diameter, 2.!6-111m. Vycor combustion tube, B , contains a semipermanent packing comprising a small amount of asbestos a t the end, 80 mm. ot coarse (pieces 3 to 5 mm. in length) cupric oside, and a small amount of asbestos which is tamped on top of the cupric oside. The remainder of tho tube accominotlates a misture of the
weighed sample and 40- to 100-mesh cupric oside. I t is convenient to have several auxiliary tubes beside the balance case for use in a series of analyses. Before they are used, freshly packed tubes should be heated a t approximately 800” C. (blast lamp) for a few minutes while t’hey are flushed with carbon dioside. No wire gauze is ever used around the removable combustion tube, but its surface should be made rough with an emery cloth, as such treatment seems to preclude star formation and cracking when the tube is placed over t,he blast lamp. Asbestos Shield. The shield, C, is plnced directly over combustion tube B. Stationary Combustion Tube. A st:indard diameter, 380-mm. T-ycor combustion tube, D,contains a permanent packing similar to Pregl’s conventional tube (6). This tube is packed as follows: A small asbestos plug 3 to 4 mm. thick is compressed into the neck of the combustion tube, followed by 130 mm. of coarse cupric oside. Another asbestos plug 2 to 3 mm. thick is inserted, followed b p . 40-nim. layer of copper vr-001 or copper wire. A t’hird asbestos plug 2 to 3 mm. thick is tamped onto the copper, followed by another layer of 80-mm. coarse cupric oside. A fourth asbestos plug 3 to 4 mm. in length concludes the permanent filling, which has a total length of 250 mm. Combustion Furnace. A conventional micro combustion furnace, E, is used. Azotometer. B conventional microazotometer, F, is used. Burners. Two blast lamps, G and G’, capable of operatin at SOO” C. are placed under combustion tube B , as indicated in h g ure 1, at the start of a combustion. (Blast lamps with burner tip 5 2 are now used, made by Anderson and Forrester Co., Ilenver, Colo.)
V O L U M E 2 3 , NO. 10, O C T O B E R 1 9 5 1
1493
Benzylirothiotma IICl
13.82
13.88 13.73 13.83
Cystine
11.66
ll.i6 11.63 11.67
3 niinutcs. G is nioved in the opposite direction from t h a t of conventional burning. The burners are removed, and the system is flushed with carbon dioxide until microbubbles appear in the azotometer. The leveling bulb is raised, and D is closed by ~iniultnneouslyturning stopcocks 3 and 4 opeu to the air. The azotometer reading is made in the customary manner after a 5-niinutc draining period. The nicthod of Pornatti (6)for removing bubbles adhering to the azotometer stem or mercury by manipulating a thin iron wire with a magnet is highly recommended. JVhile the azotonietcr is draining, D may be completely sealed by means of stopcock 2, and B may he subsequently replaced by :in alternate which contains a new snniple and which may be flushed with c:irbon dioside preparatory to another combustion.
15.12 6.18 5.88 4.57
1.5.18 6.62 5.80
DISCUSSIOh AND RESULTS
Table 1. Deteriniriatioil of Nitrogen % TImry Standard* Acetanilidc
10.37
Research samples Bromo-2-acetoxy-4-nitrotoliit~nc Calcium cyclohexyl sulfamate Calcium pantothenate Chloroaniine Diamine polymer Isoquinoline HCI
16.26 4.2?
G;
I'orind 10.33
10.38 10.37
4.65 lG.'LT, 4.16
Sto cocks. Types and arrangcnicnts of stopcocks are illust r a t e f i n Figure 1 . Rubber Connections. Parts of the apparatus are assembled h y meaw of thc uwa1 impregnated, henvy rubber tubing. PROCEDURE
The appai:ttu>, \\ ith tlie exception of the rcmovdile coiububtion tube, B, is assembled. The stationary combustion tube, U, is flushed with carbon dioxide (most conveniently by use of generator A ' ) , antl the furnace is subsequently brought to 700" to 750" C. ( I , 8). Thereafter, D and E are constantly maintained a t the elevated temperature during a series of analyses. The tube is removed from the furnace only when it becomes necessary to replace the co per packing; D contains carbon dioside a t all times, and is seared from the atmosphere by stopcocks 2 and 3 when it is not in us('.
Typical anal) tical data determined with the rapid, tworombustion-tube procedure are provided in Table I. That the eonibuqtion of halogen or sulfur compounds has a deleterious effect upon the life span of a combustion tube packing ha3 been obscrvccl :it U h o t t Laboratorieq and elsewhere. Presumably, a coating of cupric halide or sulfide IS formed n-hich renders the packing Icss effective for subsequent analyses, and many investigators burn out combustion tubes after analysis of such compounds. The use of carbon dioxide generator A' is most convenient for burning out stationary combustion tube D in a stream of carbon dio\idr, which enters the end of the tube,; impurities ale deposited i n the cold fore part of the tuhe where they do no harm. Other apparatui has been described for introducing car l ~ o ndioxide into the ends of combuvtion tubes ( 3 , & ) . The semipermanent pncking of removable combustion tube R inay generally be used for a large number of analyses; howevei, it seems advibable to replace the packing after two or three analyses if halogcn or sulfur rompounds are burned. Repacking inay be quiclrly ant1 easily performed. Carbon d i o d e generatot A ' is a convenient, but not :I mandntor); feature of the Dumas procedure employing ti! o c.ombustion tubes. It is not new-sary that the blait lamps provide a uriiform tempeiature under B from sample to sample, although a temperature of approxiniatcly 800' C. is recomI/ I A I mended. ACKNOW LEDGRIENT
F G
Figure 1 .
G'
The author i. indebted to
Apparatus
E'ol ty- to 100-mesh cupric oxide is a d d d to the semipermanent packing in B, to n height of approximately 30 mm. The samplcs (2 to 5 mg. in a boat or capillary) is dropped into the tube, the tube is rotated to mix the contents, antl :in :idditional amount (70 mm.) of the cupric oxide is added. Combustion tube B, containing the sample, is connected to d and stopcock 2, as indicated in Figure 1. Carbon dioxide is vented through B and into the atmosphere for 1 minute via stopcock 2; some idea concerning the flow of gas may be gained by directing the gas from stopcock 2 into a small beaber of ater. Stopcocks 2 and 3 are then arranged to allow carbon dio\ide to -weep through the apparatus and into the azotometer (stem open) for 1 minute. Subsequently, stopcock 1 i8 closed, the azotometer stem is filled, and its leveling bulb is lowered in ordry to apply the usual test for microbubbles. The ronibustion is started if the bubbles are satisfactorj-: etopcock 1 remains clobed. Blast lamps cf and G', operating a t approximately 800" C. ( I ) , are placed simultaneously under B prrcisely a t positions g and g', respectively. XOwire gauze is used. When the tube becomes red a t these positions, G' remains stntionary, while G is moved next to position g' slowly during 2 to
R. H. Berg, R. W. Barrori, D. S. M c C a l l u m , a n d Evelyn I-.Schuber for ninny helpful suggestions antl assistance. LITERATURE CITED (1) Brancone, L. SI., and Fulrnor, W., A N ~ LCIIEX., . 21, 1147 (1949). ( 2 ) Gonick, H., Tunnicliff, D., Peters, E., Lykken, L., and Zahn, V., ISD. ESG.CHEM.,-%SAL. ED., 17, 677 (1945). (3) Kirsten, JV., A s x . C H m r . , 19, 925 (1947). (4) Noyce, W., 16id..21,877 (1949). (5) Pornatti, R., ISD. ENG. CHmf., ASAL.ED., 18, G3 (1946). (0) Pregl-Grant, "Quantitativc Organic Microanalysis," 4th ed., PB. 68-70, Philadelphia, Rlakijton Co., 1946. ( 7 ) Ronzio, .L, IND. ENC.CHEW,ASAL.ED.,12,303 (1940). (8) Smyth, F. H., and Roberts, H. S.,J . A n . Chem. Soc., 42, 2582
(1920).
