V O L U M E 2 3 , NO. 2, F E B R U A R Y 1 9 5 1
363
10-2p. This value, although low, is significant in a vacuum system where the total pressure may easily be only one fifth of this number. Lowering the temperature of the freezing trap, therefore, is the obvious solution to prevent loss of ethane. From the above equation, the calculated vapor pressure of ethane a t -210" C. (63"K.) is 5.3 X 10-5p, which is only 0.001 of the vapor pressure a t -196" C. Experimental verification of this significant lowering of the vapor pressure is given in Table I, where the data shoiv that no loss of ethane mas observed when the condensed hydrocarbon was exposed to the pumps a t this low temperature obtained by maintaining liquid nitrogen a t a reduced pressure of 25 to 27 inches of mercury. This method (cooling a t -210" C.) has been used to recover ethane quantitatively from air mixtures containing small amounts of methane.
reported, but the amount was comparable with the experimental error of the determination. It is conceivable t'hat in the presence of large amounts of methane more of this gas niight be present in the condensed fraction with the ethane. Such contamination could be reduced by vaporizing the sample and then recondensing the ethane a t -210" C., leaving the uncondensed methane to be pumped off. Because no other refrigeralit is kno\vii which can he used to produce conveniently a temperature of approsimatelj- -210" C., liquid nitrogen a t reduced pressure s e e m to offer a unique solution to the problem of obt,aining simply the conditioiis needed for the quantitative recovery of ethanr t'roni :Lir a i d methane.
The gas mixture I{ as passed through a coiled glass trap plared in the refrigerated zone, B (Figure 2). T o effect complete removal of the condensable fraction, the gases were circulated with a Toepler pump for a t least five passes over the trap. The noncondensed gases (methane and air) were removed by exposing the system to the pumps. The amount of condensed gas was determined by warming the trap and pumping the gas into a measuring pipet identical with that described by Prescott and hlorrison (6). The measured sample was then transferred to a sample tube by circulating the gas over a 3-mm glass tube inimersed in liquid nitrogen a t approximately -210" C. The gas was scaled off in the sample tube and then introduced into a mass spectrometer where the amounts of ethane, methane, and air were determined.
The author wishes to aclinowletige the assistmcc i n the analytical work provided by Nrs. 11. E. C. Tieiles.
In all cascs the sample was found to be essentially free of methane. I n some instances a few per cent of methane was
.4CKNOWLEDGAl ENT
LITERATURE CITED
(1) Burrell and Iiohertso11, C . S. Bur. X n c a , Tech. P O ~ 142 W (1915). (2) D u m , T. H., C . Y.P a t e n t 2 , 2 1 2 , 6 8 1 (Aug. 27, 194Oj. 13) Hassler, G. L., Ihid., 2,230,593 (Feb. 4,1941). (4) H o d g m a n a n d Holmes, " H a n d b o o k of C h e i n i s t r y and Physics," 24th e d . , p. 1778, C l e v e l a n d , Ohio, Chemical Rubber Publishing Co., 1910. ( 5 ) H o r v i t z , L., U. S.P a t e n t 2,287,101 (.June 23, 1942). (6) P r e s c o t t and Morrison, IXD. ENG.C m x . , . X s . k r , , Eo., 11, 230
(1939). ( 7 ) Sanderson, R. T., U. S. I'atcnt 2,375,949 (May 15, 1945). R E C E I V E.Jul)D 11, t949
Kjeldahl Microdigestions in Sealed Tubes at 470' C. LIWREYCE RI. WHlTE ANI 3 I i R I O N C. LONG V e s t e r n Regional Research Laboratory. .Ilbany, Calif. Kjeldahl digestions for the microdetermination of heteroq clic nitrogen were carried out in heavy-walled, sealed glass tithes at 470' C. w i t h concentrated sulfuric acid and mercuric oxide cataly s t . At this temperature the digestion is complete in a fraction of the time usually reconirueiided for heteroc?clic compounds. The accuracy and precision of the method are good because there is no possibility of nitrogen loss due to thermal deconiposition of arnnioniimi bisulfate or by bumping. The pressure deleloped w ithiit the digestion tuhes is nominal.
I
S 1889 Gunning ( 5 )introduccxd the use of pota,esium sulfate to hastcri the Kjeldahl digestion by raising the boiling temperature of the digest. Its use has become general. The increased boiling temperature is especially effective in shortening the time required to obtain complete nitrogen recovery from refractory compounds. Thus Ogg and Willits ( 7 ) sholl-ed that the time required for complete digestion of nicotinic acid was approsimately halved for each IO" C. the temperature of the digest was raised, and even with as much as 625 mg. of potassium sulfate per milliliter of sulfuric acid, they found that a minimum of 3 hours was required for the complete digestion of this refractory material on the microscale. Too high a concentration of potassium sulfate has been shon 11 to cause nitrogen loss due to thermal decomposition of the animonium bisulfate formed during the digestion. Therefore, there is a practical linlit to which the digestion time may be shortened by increasing the boiling temperature of the digest through thr addition of potassium sulfate. To avoid the uncertainties incident to use of high concentrations of potassium sulfate and the long, tedious digestion required for refractory materials, the authors digest such samples with sul-
furic :Lcicl uiitl tiicsrcwric oxide in se:iled tuhw at 470' C. Gnd(>r thrw conditiolis iiitrogtin cminot be lost, through theriiial dwomposition of ammonium hisulfate or by humping. The dipstion requirvs little :ittention and it is complctd very quickly. A previously descrihrd Kjeldahl sealed-tube digestion iiiacromethod ( 6 ) ,using fumiiig sulfuric acid and requiring several hours' hcating :It 330" C., does not offer t,he advantages of a p w d and convenience inherent in thc nxthod described hpre. PROCEDURE
Weigh a 5- to 10-mg. sample into a heavy-idled borosilicate glass Carius tube ( 9 ) approsimately 7 iiiches (17.5 cm.) long. Add 40 mg. of mercuric oxide and 1.5 ml. of concentrtlted sulfuric acid and seal the tube with a gas-osygen torch. Place the sealed tube i n an inclined position on a corrugated aluniinum shelf in a welded steel box constructed to fit closely within a temperaturecontrolled muffle furnace. Close the box and insert it, into the muffle heated to 560" C. and reset the temperature control to 470" C., the desired digestion temperature. (The heat capacity of the box made it necessary to preheat the muffle to 560" C., so that t,he sample would quickly come to temperature. Under these condit,ions, the shelf that supported the tuhes reached 470"C. in approximately 15 minutes.) Heat 15 minutes at 470" C.,
364
ANALYTICAL CHEMISTRY Table I .
;inalyses of Nitrogen-Containing Compounds Nitrogen Content, % Type of Sitrogen
Conipound Cystine. N.R.S. No. 143 Acetanilide, S.R.S. No. 141 I-Lysine hydrochloride Nicotinic acid 2 2'-Bipyridine 41Pyridylpyridinium dichloride dl-Tryptophan 8-Hydroxyquinoline Hibtainine dihydrochloride Sulfnt hiazole
L-ric acid (luinine aulfate
()uinidine Fulfate
R trych nine sulfate Brucine Yulfate .$tropine sulfate 4-Kitroacetanilide
-4mini,pyrinv
Amine Amide
Average Replicates 11.66 1 1 . 6 1 11.63,11.63,11.60,11.60,11.59 10.36 10.32 10.34, 10.32, 10.32, 10.31, 10.30,10.30 15.34" 1 5 . 4 1 1 5 . 4 4 , l 5 , 4 0 , 1 5 . 3 9 11.42 11.46, 11.43,11.42,11.40,11.37 11 .38 17.87 17.92,17.85,17.84 17.94 12.11 12.21,12.10, 12.09,12.05 12.23
Theory
Aniine Pyridinr Pyridine Pyridiniurn and pyridine Indole a n d 13.72b amine Quinoline 9.65 Diazqle a n d 2 2 . 8 3 anme Thiaz,ole. 16.46 amine and amide Purine 33.33" Quinoline 7.50 bicyrloand I-amoctane Quinoline 7 . dO and 1-alaoctane bicycloIndole or dipyrrole Indole or dipyrrole Nortropane Nitro and amide
Pyrazplone and
13.67
1 3 . 6 8 , 13.66
9 . 5 6 9 , 5 S f 9 . 5 7 , 9 .2 2 22.86 2 2 . 9 1 , 2 2 . 8 6 , 2 2 . 8 2 16.35
16.39,16.33,lfi.32
33.13 7.45
3 3 . 2 0 , 3 3 . 1 6 , 3 3 . 1 1 , 3 303 7.49,7.44,7,42
7.51
7 . 5 3 , 7 . 5 1 , i ,JO
7.31
7.25 7.26,7.2:, 7.23
6.31
6.23 6.26,6.22,6,11
4.14 18.66
4.14 4.14,4.14,4.14 1 1 . 1 5 1 1 , 1 6 , 1 1 .1 5 , 1 1 , 1 4
18.17
1 4 . 7 2 14.83, 1 4 . 7 6 , 1 4 . 5 8
amine Sitrugen content by routine Kjeldahl nricroniethud with: a 110-niin. digestion, 15.40% ( 1 2 ) . b 360-niin. digestion, 13.70% (11). C 80-min. digestion, 33.14%. -
~~
__-.
~~.~
._.___.
~
remove the box from the muffle, and allow it to cool slowly or place it under a multiple-jet air blast to cool it quickly. After the box reaches room temperature, remove the tube and open it a t temperature as previously described (9). Dilute the contents of the tube with 2 to 3 ml. of water and transfer it to a 50ml. beaker with 8 to 10 ml. of water. When COO^, wash the Sample into the Kjeldahl still containing 8 ml. of a solution of 40% w./v. sodium hydroxide and 5% w./v. sodium thiosulfate. Distill and titrate the ammonia by any standard procedure. RESULTS
The analyses of Satiorial Bureau of Standards cystine and wet'anilide, lysinr, a group of heterocyclic nitrogen compounds, and representative samples requiritlg reduction before the Weldah1 digestion are presented iii T:1k)lc I. blany of the compounds listed have not been reported to 1)e refract'ory,but they have been included in this study t o show thitt the metshod is effective for a xxriety of nitrogen-coli t,ainirigrings. Tryptophan and lysine are among the compounds usually reyarded as being refractory. The results for these amino acids are in good tvith theory and lvith the values previously found for these samples (11)by routine Kjeldahl microdeterminations nit,h extended digestion. The refractory nature of nicotinic acid is probably due to the presence of nitrogen in the heterocyclic pyridine ring. Pyridimt y p nitrogen was determined satisfactorily by the present method in nicotinic acid, 2,2'-bipyridine, and 4-pyridylpyridinium dichloride. Compounds containing a variety of other heterocyclic nitrogen structures were analyzed successfully (see Table I). .\mine and amide nitrogen were also recovered quantitatively. Clark ( 2 ) reported that atropine and quinine required extended digestion, and Drevon and Roussin (3)found that quinine was not properly analyzed under their conditions. Severtheless, thest. and the other alkaloids listed in Table I rvere analyzed without difficulty by the met,hod described herein. The data for 4nitroacetanilidr and aminopyrine show that this lllcthod cannot be used in place trf t.he Friedrich hydriodic acid
procedure ( 2 , 4, 8) for nitrogen linkages requiring reduction prior to the usual Kjeldahl digestion. The data in Table I1 show that the amounts of acid and cat,alyst used are not critical. Thc amounts specifird in the procedure were chosen so that the same reagent dispensers could be ustd for this and for the Clark ( 2 , 10) method routinely used in this laboratory. The data in this table also show that the digestion is complete after 7 niinutt'q of heating a t 470" C., n-hen mercuric oxide is used as catalyst, but that 30 minutes are required i t i the absence of catalyst. 4 15-minute digestion with catalyst w t b chwcii to provide an ample margin of safety. DlSCUSSION
The relatively high temperature eniployed, about 120" C. higher than that pract,icable in thv open flask, permits the rapid and complete digestion of refractory compounds without. loss of any free ammonia tvhich might be generated by dissociat,ion of ammonium bisulfate. The met,hod should also be useful for the analysis of volatilt. samples or those t,hat form volatile nitrogenour: products during digestion. The complete digestion of the sample after 30 minutes' heating a t 470" C. without catalyst, (Table 11) suggests that this method should be useful when other constituents (sodium, potassium, phosphorus, etc. must be determined in addition to nitrogen. The sealed-tube method of digestion rtquires no new techniques, because the sealing and opening of the tubes are the same as for the Carius microprocedures and the distillation is the same as that usually used, except +,hat the base must be added to the still before the sample. This is rcquired because the carbon dioxide, sulfur dioxide, and other acid gases formed during the digestion are not driven off as th during the open-flask digestion. '4ddition of the base still prior t o the sample is recommended for the convrntioriiil procedure by Belcher and Godbert (1j. The welded steel box was used to permit the digestion of several samples at' a time and as a safety shield. The corrugat,ed shelf that supports the tubes was lnadc of aluminum t o provide. rapid heat transfer to the tubes and to promote even ttxn1prraturr throughout the box, In preliminnry work the tubes Lvcrc heateti in 8-inch lengths of 0.5-inch black iron pipe provided 1vit.h two Screlv caps and a small vent hole n(>itrthr top.
Table 11. Effect of Length of Digestion and Amounts of Reagents on Reco\veryof Nitrogen from Nicotinic &id Nitrogen Found
Tinie Digested a t 470' C.=
Sulfuric Acid
Min.
341.
M g.
1
0.5 0.5 1.5 1.5
13 0 40 0
0.5 0.5 1.5 1.5 0.5 0.5 1.5 1.5 0.5 0.5 1.5 1.5
13 0 40 0 13 0 40
1.5
30
a
Mercuric Oxide
0 13 0 40 0
R of theoretical
% 9.42, 2.53, 7.66, 4.40,
8.96 2.83
8.52 3.94
11.38, 1 1 . 4 3 9.85, 9 . 7 3 11.37, 1 1 . 4 1 8.63. 8.81 11.38,11.43 9.85, 10.63 11.39 11.41 10.18: 9 . 7 7 11.39,11.39 11.29,11.48 11.44, 11.41 11.35,11.33
82.8, 22.2, 67.3, 38.7,
78.7 24.9 74.9 34.6
100.0, 100.4 86.6, 85.3 99.9. 100.3 76.8, 7 7 . 4 100.0,100.4 86.6, 9 3 . 4 100.1,100.3 89.5, 85.9 100.1,100.1 99.2, 100.9 100.5,100.3 99.7, 9 9 . h
Temperature of shelf t h a t supported tubes. ~
... .. .
V O L U M E 23, NO. 2, F E B R U A R Y 1 9 5 1 The pressure resulting from the digestion of the 5- t o IO-mg. samples specified should amount to only 8 few atmospheres. That the heavy-walled tubes used are amply strong t o withstand the pressure is demonstrated by the fact that not a single tube failed a t any time during this investigation. I n order t o test the tubes further, 50-mg. samples of dextrose and 33-mg. samples of corn oil were digested a t temperatures up t o 525" C. for several hours. Thin is 55" C. above the temperature specified for the method and 15" C . above the strain point of the glass used. Samples of this size a t the higher temperature generate very much more pressure than do the 5- t o IO-mg. samples a t the temperature used in the method. Even under these extreme condit.ions, there was still no failure of the tubes. As 8. safety precaution, commercid Carius miorotubes should not be used for this procodurc and t.he tubes should come t o room tcmperature before they are removed from the shield and opened. If desired, the digestion box can bo opened behind a safety glass shield, and the pressum within the tubes can be released before they are removed from the hox by applying a small, sharp flame to the tips of the tubes until they open as a result of the slight internal pressure. The results obtained nith this met.hod show excellent precision
365 and accuracy, and bot.h umking and elapsed times are very favorable when compared with the long digestion procedures often recommended for hctcrocyclic or refmotor? nit.rogcn compounds. LITERATURE CITED
(1) Belohdr, R., and Godbert, A. L., "Semi-Micro Quantitative Organio Analyais." P. 91. London, Longmans, Green and Co.. 1945. ( 2 ) Clark. E. P.. 1.Assoe. Oi% Am. Chemists, 24, 641 (1941). (3) Drevon, B., and Roussin. J . pham. chim., (9) 1, 24 (1940). (4) Friedrieh, A,. Kiihaas. E., and Schniirch. R., Z. physiol. Cham.. 216,68 (1933). (5) Gunning, J. W., 2. anal. Chem., 28, 188 (1889). (6) Levi, T. G., and Gimignani, L., Cess. chirn. ital.. 59, 757 (1929). (7) .~ Ogg, C. L.. and Willits. C. 0.. J. Assoc. Ofic.Am. ChsmbFta.. 33.. 100 (1950). (8) Seoor, G. E.. Long, M. C., Kilpatriek, M. D.. and White, L. M.. Ibid.,33. 872 (1950). (9) White, L. M., and Seoor, G. E.. ANAL.CEIBM.,22, 1047 (1950). (10) White. L. M., nndSecor, G. E., IN". ENG.CHEM..ANAL ED..18, 457 (1946). (11) White, L. M., Seeor, G. E., m d Long, M. D. C., J. Assoc. OBc. Ag7. Chcmists,31. GSi (194X).
Microscopic Fusion Analysis of Sterols VICTOR GILPINL, Yale University, New Haven, Conn.
I-
move8 rapidly into melt. During the coolA recently d e v e l o p e d ing period, the crystallization velocity abmethod of miemsoopio has been shown in the identification of ruptly decremes and parallel or curving identification has been incompounds by means of microscopic fusion blsdos and rods appesr; meanwhile, the erysvestigated by which mPthods. Although all such methods depend tal front changes from smooth to serrated. minute quantities nf on the same phenomena, two differing techOn further cooling, the crystallization velocity sterols can he easily a n d niqucs have been developed. For convenincreases slightly. Transverse shrinkagr rapidly recognized. Aliencc, these may be called the. Kofler method, cracks appear soon after complete solidifics, t h o u g h there are limitain which a hot stage is used (3), and the Mction. tions to the method, it Crone method, in which a hot stage is not Partially Remelting Solidified Melt ("Meltshould pmve a valuahle u a d ( 1 , 4). The latter method has the adback"). Bladelike crystals grow (Figure 1). a i d i n the field of sterol vantaees of m a t e r soeed and simnlieitv. . - AlSolidified Preaaration. The microcrvPtalanalysis. mo& all the data on compounds studied by line solid often shows a herringbone pattern t,he McCrone technique appear as a part of (Figure I). Bladelike crystals show nearly x large cryst$lagraphic program (6). It is parallel extinction, negative elongat,ion, low birefringence. thv aim of the present study t o explore the possibilities of the and newly eentored O.A. or off-oenter Bz. (optic sign positive, McCrone analysis as an independent means of identification of 2V large). n h t e d compounds. The sterols are excellent materials for this m r k , because they melt a t moderate temperatures, usually without decomposition. Y RECENT years, considerable interest
~~
To describe interference figures where no ,optic axis is visible the following terms have been employed: Indefinite-for relatively clear figures, from single crystals, who? orientation cannpt he definitely specified; one brush-for relativcly clear figurcs in which one isogyre sweeps the field on rotation the stage; diffuso-for a. conoscopie view consisting of supenmposed figures, the result of several crystals in the field a t once. Not all observations yield useful data for all compounds-for example, heating some compounds gives rise to no sublimation, decomposition, or mesomorphs ("liquid crystals"); in such cases, observations on heating the solid are omitted. Again, if the erystdlilliaation velocity of the solidifying melt change with temperature is not reported, it is t o be inferred that such change is slight. Omission of the optic sign and estimate of 2V from interference figure descriptions implies that the figure8.a~not sharp enough for reliable determination of these properties. All conoscopic observations were made with a 0.85 numerical aperture dry objective; all photographs and observations (except refmct,ive index) were made with crossed Nieals. CHOLESTEROL
Cooling and 'Solidification of Melt. The melt su~ercoals slightly. A microcrystalline mass solidifies spontaneously and L Present address. Department of Chemistry. Michigan State College. East Lansing. Mieh.
Figure 1. Change i n Crystal Habit duringSolidification of Cholesterol Mixed Fusion of Compound with Thymol. Iilrtdev and needles st boundary show poor profile angles; one refractive index equals melt, and one refract.ivc index is greater than melt,. After a few minut,es, nuclei of a new phase, possibly an addition compound, appear at the boundary. The bladelike crystals of thi8
