1298
ANALYTICAL CHEMISTRY
Table 11.
Summary of Results on All Synthetic Mixtures
R. H. Kienle for making available to them the facilities of the Calco Chemical Division, American Cyanamid Co., for thc experimental work. L I T E R 4 r U R E CITED
40 0 47 0 c13 0 60 0
40 1 46 8 53 i 60 0
40 16 46 9 4 52 80 60 5.5
0 0 0 0
4Q
81 80 66
n
17
0 16 0 20
Allen, Eugene, 1'h.D. thesis, Rutgers University, 1952. Bastian, R.,. ~ - A L . C H t x . . 21, S i 2 (1949).
0 21
Ibid.. 23, 580 (1951).
the t,ransniittancc rr:adings uscd in det'crmining the data for the calibratiori are in error by +0,5%) the results obtained in an analysis by the conventional method will be in error by =t4.8yo, whereas the error incurred in the prcfert,nco nirthod will be 2~1.4%. ACKNOWLEDGMENT
The authors wish to acknowledge the valuable suggestions of C. F. Hiskey and Rohert Bastian. They are also indebt,ed to
Bastian, R., JYebwling. It., a i d I'dilla, F,, Ibid.. 22, 160 (1950). Hiskey, C. IT., I h i d . , 21, 1440 (1949). Hiskey. C . F.. and Firestone, D., Ihid.,24, 342 (195%). Hiskey, C. F..Rahinowitz. J., and Young, I. (2.. Ihid.. 2 2 , 1464 (1950). Stearns. E. I., i n .\Icllon, 51. (;., "hnalytical Xhsorption Spectroscopy," pp. 352-5, 309-i3. New Tork, John \Viley & Sons, 1950. I-oung, I. G., and Hiskey, C , F.,. ~ - A L .CHEM.,23, 506 (1931). RECEIX-ED for review S o r e m l x r 6 , 1951. Accepted I p r i l 23. 19.52. Presented in part at the .\ieeting-in-3Iiniatiire of the N o r t h ,Jeriey dcction, . t \ I E R I L . * \ . CHE\IICL ~ O C I R T I . , X e w s r k . N..I., .January 9, 1950.
Chemical Analysis and Isotopic Assay of Organic Compounds R . CHRISTIAN ANDERSON, YVETTE DELABAHKE,
AND AKSEL A . BOTHNER-BY C h e m i s t r y D e p a r t m e n t , Brookhaven .\-ational Laboratory, C'pton, Long Island, Y.
The present work was begun to provide a method for the concomitant chemical analysis and isotopic assay of micro samples of labeled organic compounds. The possibility of serious errors in obtaining representative assay results requires a complete interlocking of both analysis in the classical sense and the isotopic assay. A method has been developed which w-ill give, by use of a single niicrosample, carbon and hydrogen percentages and, in addition, deriiatives that can be used for C13 or C" and deuterium assay. The precision of the chemical anal) ses equals that of the standard Pregl method, while the assay procedures are as precise as any now available. The simplicity, rapidity, and accuracv of these methods will make them extremely valuable for workers in isotopic research and for analysts in general.
N
UMEROCS methods have Iieen devised for the isotopic
assay of carbon and hydrogen in labeled organic compounds. The usual procedure involves the combustion of the organic material to carbon dioside and xater, which are the most practical derivatives for accurate and reproducible results-carbon dioxide for carbon 13 assay, cnarbon dioside or barium carbonate for carbon 14 assay, and R-ater, ethane, or hydrogen (as secontiarj. With a majority of the comderivatives) for deute ljustion methods the e as been on the preparation of the ., and the problem of the associderivatives for the is0 ated chemical analysis has been relatively untouched. It is not desirable to do combustions without analytical control, inasmuch as the rates of combustion of different carbons in a given molecule vary widely, and the carbon dioside produced is not representative until the sample is completely burned. The serious errors t h a t may arise in this manner have been noted by Armstrong and his coworkers ( 1 ) . Some methods have inherent 7 .
.\-.
errors, while others are not suited to quantitative or uncomplicated techniques. For example, methods in which the combustion carbon dioside is led into barium hydroxide solution as it is being produced, will give an inhoniogeneous barium rarbonatr precipitate in respect to uniform specific activity. This, coupled with the ubiquit,ous self-absorption and scattering phenomena associated with weak beta emitters, makes for a situation too comple\; to appraise or control. Finally, it is cust,omary for the organic. chemist to provide evidence of the purit>- of his compounds by obtaining acreptable analyses for both carbon and hydrogen, which usually are provided only b!. a dry com\~ustionmethod. The method described here attempts to be simple and acrurate, to provide t,he masimuni amount of information per sample, to avoid difficult, manipulation?, and to be flesihle enough to allow use of the derivatives in equipment that ir widtlly 01' c,asily available. The combustion method is based on that desc*i,il)c,l l i Saughton ~ and Frodyma ( 7 ) . APPARATUS
Figure 1 shows t h r various components of the c~omt)ustion train in relation to each other, prior to assembly. It w;ts found feasible to join certain parts bj- st:rndard-taper joints sealed with Apiezon W wax and the others 13). direct glass-to-glasfi juncations. Thus the components that involve the combustion tulw ('ail lie dismantled quickly for repair, cleaning, or replacement, whereas the units comprising the traps, calibrated volume, and manometer are permanently sealed because very little, if any, servlcsing is required. The oxygen is cleaned and controlled by conventional mraus: cleaned by passage through a drying tower containing alternate layers of Anhpdrone and. A4scariteseparated by glass wool, and controlled by a reduction valve directly on the gas cylinder in addition to a glass regulator using concentrated sulfuric acid. The oxygen is then passed through a preheater as shown in Figure 1, which resembles the one described by Niederl and Kiederl (8). From there the oxygen is passed through a U-shaped
V O L U M E 2 4 , N O . 8, A U G U S T 1 9 5 2
1299
trali equipprd with a 2-11111i. hollo\v plug sopcock oii tlie coiiiburtioil tulw sitic, :itid ti\-o 12/30 '$ joints a t each end. T h r liody of i h e trap i. made of boro ilicate glass tubing 13 min. in outside iIi:iiiieter, with a 14 '35 taiidard-taper outer joint for ase this stopcock as well iezoii S grease is applied
Tlir: c.ointju&on tulie is made entirely of quartz, iiicludiiig the t l i i w et:~ndarti-taperjoints and cap. The body of the tube is 13 n i i i i . iii outside diameter and 45 cm. long with a constriction iieat' t1ic outlet (,lid to hold the filliiig. T h e joints a t the inlet :iiicl outlet end a r e 12 :30. ~vhile tlie sniiiplr eritraiice vonsists oi :i 11 35 iriiier joint, ~ i t l ai cap Ivhich is miitici so :i$ to reduce its iiitei,ii:tl voluine to a iiiiriiinu~n. Lug. tiw 1)i.ovitiecl so that it npriiig c:iii lie usec! i o hold the c a p i i i pl:ii*c. 'The outlet eud 01' [lie lube can iie t w i t as ~ho\\-iito :ic~c~oiiiiiiod:itt~ tiie triiiii (Figure 1) or tlie heavl- wtitvi' t i x p (Figtui,cj 2 ) : o r Iri't stmig i t ror use jvith tiit3 nitrogen oxide i,duc$ei,(I*'igui,r2). Tliir I i c ~ t i t iiii the t u l x rhoriltl IJC1ot:ttecl appr.osiniatcly 15" i ' i ~ ) i nt i l t . inlet t i l l e. so 11i:it t h e lattt'r i. :i\\-:ij- from the :tii:ily.~trlut,iiig
1 liwc tliffere~irj , i r w x oI' app;iuiius raii 1)cx used t o coliiicrt t com1,ustioii tul)v t o thv trap rystcJni, clcpeuding u n t h c i'tl 1uirc.merits. , 7
I1 tlie -;iuiple ,,oiii:iiiiF i,arlioii, Ii~-di,ogen. 18 01' 14 liut I I O iiitrogeri. tlie train by 1 ) . Tliia is merely a coiinertiiig t u l x iixide 13 miii. iii outside diameter with witable joints. I t is sealed t o tlie systeni n i t h Apiezoii tilack \\as, aiid requires 110 further Littrutioii. If thc compound coiitaiiia deuterium, the heavy e Figure 2) is used instead of the bypass. This atid outlet joints identical in size arid position SP PO t h a t thcsr two pierep can he iiitei~changed \\illrout tli~turlririgthe i v t of the :ilii)aratus. T h e heavy \v:iter i::ii,I)oii
trap made of t u h g 8 inin. in outside diameter is 10 cm. long l)elow the 2-inm. Iiolloiv-plug st,opcocks. 4 bulb of about 3-nil. rapacity is placed a t the bottom, and the trap is narrow enough 011 the bottom half to fit into a small Dewar, and wide enough to :ic~comiiiocl:itetho stopcocks a t t h e top. This trap is affixed 1 0 the apparatus with r2piezon S bvax for ready removal. But iiiasniuch as the coiii1~ustioiitube outlrt is at a slightly cl(1v:ited ternl)erature, the joints must t i e carefullv inspected to 110 sti,i;itions appear iii the grease, indicatiiig leakage. e1iiniii:ite a11 uriiiecensary sources of Irakage in the systeiii that the tiypiss sealed on I\.4piezoii l~lack \v:ts is used when deuterium is not being iyed. If t h o water is to 1)e useti directly l o r assay tiy the falling d i v i i method, the trap is modified so that oiie of the stopcocks is r e p l a r d 11). ii hoilo~v-plug. right-angle vacuum stopcoc*k (similar. to Corning stopc'ock S o . 7.548) \vhich : ~ l l o wfor addition of solids t o the tulie 01' r(movii1 of the \v;i~tbi. Ivith a pipet. T h t , iiitrogeri oxide reducer is e i n p l o ~ ~ ewhen d the c,oinpouiitl to lie ;irialyzed coiitains nitrogen. I t consists, from inlet to outlet end. of a 12/30 T joint to fit the coml)ustiori tube; a 2-mm. hollon plug Ptopcock (SB. Figuw 2), a those tlescrilwd tielow under the trap three-way double-oblique, I ~ o r cstopcock, which leads iiirectl,~to :iiiotlier 12,30 joint for connectioii to t h r t r a p sy.st(xiii. T h e upper arm of the three-way stopcock ends iii ;I 12 4 0 joiiit. To this is affixed a quartz section :is sho\m, the lieiiig mni. i n outritlc iliameter atid t l i r outel' tuiw inlier t u i ) ~ 1 5 iiitii. T h e outlet entlr in another 12/80 joint tvliich fits into :i 1)orosilic.titp glass joint conriectetl to the loiver arm of th(J t i i i w - w : i ~ - *iopcoc.k via a 2-111111. straight-!)ore Ptol)co(.k. S8. The aiiiiular s p a i ~in tlie quartz tr:ip is filled with R roll of finclmesh copliei' wreeii. f r e ~ h l yretiucutl ivith nic~th:arioi, I)efor,e the trap is se:tled off a t the top. I t has l i e e n t'ouild t:spedierit to sei u p t\vo separate coiiiliustioii traiiis.. one for. riitrogrii-c:oiit:tiiiiiig sul)stuticcs i i i i d the other for coinpouiids free of Th(L reducer \rorks in the follo\ving way. T h e carhon dioxide. water. and the riitrogeii o s i d w evacu:tted system by niariipulatiug stopcock gases except osygen are caught in the spir:il trap c,ooletl with liquid njtwgen. a f t e r the coiiiljustion is complete, the excess osygeri is pumpcti off, :ind the g w e s are pumped through t h r quartz tulw c*ontaiiiingcopper a t 450' C. (see below) where the oxides of iiitrogeii :ire reduced. Thrl csrlion dioxide and \vatel :tre theii trapped in the regular \ T h e trap section is made of t u 13 mm. in outside diametcr :ind consists of, starting from the inlet end: a 12/30 inner joint: :I 2-mm. hollow plug stopcock, 5'2: a spiral trap 15 cm. long. i\-ith $1 IO-nil. 1)ulh a t the tiottom. t h c s pliirnls being :tI)out 8 min. aDart; a 2-nini. Tliore three-n-ay stopcock 5 cni. from tilt, traps on either side; :mother trap as described above; arid tinally a 2-mni. hollow plug stopcock, 83, the outlet end of \vhich is coriiiected to the t i a c k - d if f u sion trap dercribetl lJt'lO\+,
s5
M
TWO-LIQUID MANOMETER CONSTRUCTION
a"
=y=-
52
vAc
J
PRE-HEATER
ABSORPTION
COMBUSTIOh TUBE
T
M
H 2 0 3 P
COz T9AP
By-ppss
Figure 1. Combustion and Gas Measurement System
LJ
BACK DIFFUSION TRAP
The trau section is c o n n e c t d d via the tliree-way stopcock t o the gas measureiiient section, which cmonaists of two parts; the calibrated volumes, marked 1'1 arid in Figure 1. anti the two-liquid manoTeter. The calibrated volume has two bulbs, I:oth 45 mm. in dianieter; TTl is 80 mni. long and by2 is 180 mm. long, connected I)? a 2-mm. stopcock, S4. Two outlets are provided on VI, one of which is tubing 10 mm. in outside diameter to be sealed to the manometer or.
ANALYTICAL CHEMISTRY
1300 preferably, having an 18/9 spherical joint, and the other consists of a 13-mm. tube with a 2-mm. stopcock, S5, furnished with a 12/30 outer joint to which the carbon dioxide collector tube or counting tube can be affixed.
If a greater range in the size of samples is desirable, a larger number of bulbs could be provided and a stopcock system devised to increase the flesibility of the system. In actual practice, the 2-bulb section has proved adequate] inasmuch as some control over the sample size can be exercised by weighing. This system can handle sample$ that contain from 0.3 to 6 mg. of carbon.
The oil was chosen because its viscosity is lower than that of the diffusion pump oils, such as Octoil, used previously. This is important because a finite time is required for the oil to drain in the capillary from the position given by the measured gas back to the original point. By measuring t'he zero value in the nianometer both before and after a given reading, variations due to temperature changes, if an)-, can be duly noted. The only requirements for the oil employed are low viscosit,y and very low vapor prespure; consequently, oils sther t'han the one used here may t i e as good or better.
HEAVY WATER TRAP
f
L J
NITROGEN OXIDE
cop COUNTER Figure 2.
When a vacuum of 1 X 10-4 mm. is reached, the mercury is distilled into the U-tube until it com letelv fills the tube and about 2 cm. of the lower two bulbs. Tgis distilling flask, Hg, is then sealed off and the constriction just below this flask is also sealed x-ithout removing the supporting tube. The oil is now distilled in and allowed to fill the loxer right-hand bulb and 1 to 2 em. of the capillary. The whole apparatus is let cool to room teniperature. If the oil meniscus still appears in the capillary to a height of no more than 3 cm. and the vacuum remains at 1 X mm., the system is sealed off as shown. If done correctly the manometer will be trouble-free, and requires no further attention. Obviously, however, if too much pressure is applied a t the inlet end, gas will be blown into the side containing the oil and the manometer is of no further use. The manometer can be waled directly to the outlet provided on 1-1 (31 to V), which requires rare in vien of the above statement, or it can be connected by a spherical joint (1817) previously supplied at the inlet end and capped for the filling operation. The use of a spherical joint also facilitates vertical alignment of the manometer capillary.
Accessorj Equipment for Analysis and .Assay
The ho-liquid inanonietcr is a critical part of the apparatus and, as such, should be made with some care. The right-hand drawing in Figure 1 shons the manometer as it originally appears] and the other as it appears after being filled and w t h 1 atmosphere of pressure on the inlet end, In operation a t a f e x centimeters pressure, the mercury (dark hatch) is about equally distributed in both the lower bulbs and in the extended U-tube, while the meniscus of the oil is somexhere in the capillary section. From the bottom up, the manometer dimensions are as follows: The lower U-tube is made of heavy-walled tubing 10 mm. in outside diameter, and is 22 em. from the bottom to the beginning of the cylindrical bulbs. These bulbs are 37 mm. in outside diameter and 60 mm. long. T o one bulb are affixed two sections of tubing 10 mm. in outside diameter] one for the ultimate connection to the calibrated volume, the other, temporary, to the 50 ml. distilling flask, Hg,on the left and t o the spherical joint at the very top of the manometer. There are constrictions as shown to facilitate sealing off the various glass portions in vacuo. To the other bulb is connected a 50-cm. length of 1-mm. capillary. At the top of the capillary is another bulb, also 37 mm. in outside diameter and 60 mm. long, which has attached a tubing 10 mm. in outside diameter leading to the spherical joint for evacuation and to a 50-ml. distilling flask, 0. Before filling, the manometer was treated with cleaning solution, rinsed with distilled water, steamed for 1 hour, and dried overnight a t 130" C. T o fill, the whole apparatus is connected to a high vacuum manifold a t the spherical joint. Clean mercury is placed in flask Hg and dibutoxytetraethylene glycol is placed in flask 0. Both flasks are then sealed off from the air, and the inlet tube, XIis sealed off or, if with joint, plugged. The manometer is evacuated and carefully baked by use of a soft flame, and the oil is outgassed by cautiously freezing and thaiTing.
To determine the position of the oil meniscus in the nimometer capillary, a special centimeter scale, equipped with a sliding vernier, is fixed parallel to the manometer. The scale consists of a milk glass strip on which are ruled fine I)lack lines 1 mm. apart running from 0 to 50 em. Ever>- fifth and tenth line is extended in the usual way to aid in reading. The vernier is scribed on a clear glass plate 3 1 8 inch thick with parallel lines on both faces of the plate coinciding with the zero line of the vernier. This arrangement eliminates parallax even with the manometer 5 mm. behind this plate. It is thus posil)le to measure directly the height to which the oil rises to 0.1 nini. for each reading. Inasmuch as the average rise in pressure is usually more than 10 cm., the error per reading is 0.1%. This represents the general order of precision obtained with the present lipparatus. The hack-diffusion trap is connected into the a paratus betv-een the stopcock and the mechanical pump whic1 is used for evacuation of the whole system. It has a 29/42 joint which epta the trap body, 27 mm. in outside diameter I)>- 14 ?in. long. which can be removed for periodic cleaning. Any mechanical punip that can give a vacuum of 0.005 mm. under operat,ing conditions is satisfactory. Higher vacuums are not only unnecessary but. undesirable. Use of a diffusion punip backed by a mechanical pump caused the analyses t'o be very erratic, possibly because the glass surfaces of the apparatus were desorbing gases in some unreproducible manner undrr these renditions. PROCEDURE
The apparatus is condit,ioned for use in the follorving niiiniier : The two combustion furnaces and the preheater furnave are turned on. Stopcocks S 1 and S5 are closed, and stopcocks S2, 83, and S4 are opened, while the T-bore stopcock is in position as shown in Figure 1. T h e back-diffusion trap is immersed in liquid nitrogen and the mechanical pump is turned on. A4fter several minutes the oil level in the manometer is noted. Stopcock S2 is t,hen temporarily closed as S1 is opened to admit 1 atmosphere of oxygen into the combustion tube. Then S 2 is opened slightly to bleed osygen into the evacuated trapmanometer section, the rate of flow- being adjusted to give a rise in pressure on the manometer of 3 cm.; the manometer thus doubles as a flowmeter. The oxygen is passed through the syetem for 15 minutes, after which time the apparatus is ready for analysis. A blank can be run a t this time by merely immersing the water trap in a slush of acetone-dry ice (the T o is kept below -65' C.) and the carbon dioxide trap in liquid nitrogen and allowing the oxygen flow to continue for about 10 minutes (the standard analytical procedure time). After this, S2 is completely closed
V O L U M E 2 4 , NO. 8, A U G U S T 1 9 5 2 and the system is pumped down to the pressure originally indicated on the manometer. Stopcock S3 is closed and the T-bore stopcock is burned to connect only the carbon dioxide trap with VI and the manometer, S4 being closed. T h e liquid nitrogen bath is replaced by a beaker of water a t room temperature and the trap is allowed to come to equilibrium. T h e manometer reading is noted once more. I n a properly operating system no change can be determined. Similarly, the blank for water, determined in an analogous manner, should also be zero. An appreciable blank results either from impure oxygen or from a leak in the system, and must be eliminated before an analysis is undertaken.
For the analysis of compounds containing carbon, hydrogen. atid oxygen, b u t no nitrogen, the weighed sample, in a platinum boat. is introduced into the combustion tube with oxygen flowing out of the inlet tube. T h e tuhe cap, sparingly greased with Apiezon S , is put on and held in place with a stainless steel spring. T h e oxygen flow is maintained a t a 3-Cm. pressure on the manometer t~ the right of S2 and the traps are immersed in the proper cooling baths. The sample furnace is then pulled over the cornbustion tube and moved down finally to cover the sample; this requires a total of 5 minutes. At the end of this first combustion S1 is closed and S2 slo~vlyopened to keep the flow a t 3-cni. pressure until an almost complete evacuation of the combustion tube occurs. Then S1 is opened to allow a 3-cni. manometer pressure sweep of the combustion tube with the furnace over the sample boat, S2 being open: the operation of this second combustion requires 2 minutes. These times are usually measured by a stopwatch and noted, especially in the case of new compounds. S1 is then clused, and the system is evacuated completely until the first position in the manometer is re-established. This usually requires 3 to 5 minutes. S2 and S3 are closed and the T-hore stopcock is turned to connect the T'2 and the manometer, as carbon dioxide trap with T'i or Vl tiescritled above for the blank determination. T h e cooling bath (liquid nitrogen) is replaced by a beaker of water a t room temperature and the rise in the manometer is noted on reaching equilibrium. JVhen the determination is made, the carbon dioside is pumped away by opening S3 (see the procedure for isotopic assay of the carbon dioxide below). When the manometer comes back to the zero reading, the T-bore is turned to connect the water trap to VI or 171 T'p and the manometer. ThP leading for water is ascertained. -4gain after the determination, the water vapor is pumped away through the T-bore stoprocak and S3, thus making the apparatus ready for the next determination. For the analysis of conipounds containing nitrogen, the nitrogen oxide reducer system is put in the apparatus in place of the train bypass. T h e reducer as shoil-n v a s designed for a separate train, identical with that shown in Figure 1, escept that the connecting stopcocks are horizontal to accommodate the reducer. I n this procedure stopcock S6 assumes the function of stopcock 5 2 in the non-nitrogenous apparatus, and 8 6 is used to control the flow into the spiral trap which follow immediately. .4ll the gases from the combustion except oxygen are trapped here, while following the burning and evacuation procedure as outlined. T h e oxygen is pumped away by keeping stopcock Si in the position shown in Figure 2. so that i t does not oxidize the hot copper gauze. JVhen the combustion is complete and the oxygen excess evacuated, Si is turned t o connect this quartz tube and heated t o 650" C. by an electrical resistance furnace with the spiral trap and S8 is now opened very slowly while the cooling bath is removed from the reducer trap. The carbon dioxide and water pass unchanged through the hot copper arid are trapped in their respective places, while the oxides of nitrogen are reduced to nitrogen, which is pumped away. The carbon dioxide and water are measured in the usual manner.
+
+
S o difficulty was found in assaying for deuterium in compounds containing nitrogen. In these cases, the water was condensed in the heavy water trap and the carbon dioxide and nitrogen oxides were condensed in the reducer trap between S 6 and Si. T h e subsequent procedure is as above. The water so obtained had very little, if any, nitrogen oxides as contaminants. What might have appeared \ms removed in the operation of convert,ing the x a t e r hydrogen to ethane hydrogen and the assay of the ethane in a mass spectrometer. Presumably, the purificat,ion methods employed t o prepare the water for the falling-drop technique would also eliminate any error from this source. The apparatus is calibrated by burning National Bureau of
1301 Standards sample 140 benzoic acid and noting tlie rise in pressure of carbon dioxide and x a t e r when various amounts are employed. In the apparatus shown, using a series of six combustions, the rise in centimeters per milligram of carbon was 6.17 i 0.016 and the rise in centimeters per milligram of hydrogen was 35.85 i 0.17. It is possible to calibrate the volume of the trap 8)-stem by at,taching a carefully measured volume, Vy>a t the carbon dioxide takeoff and evacuate it along with the rest of the s>-steni prior to combustion. By then noting the ch:inge in pre.%ure going from the standard volume including T7, or 1'1 f F-2 to that volume plus Trri and making a correction for the volume in the carbon dioxide take-off tube, it is possible to determine the trap vblunie. Then knoning the density of the manometel, oil and the ratio of cross seetioris of the manometer. an independent (.heck on the validity of the empirical calibration with benzoic acid can be made. In addition, a mass spectrometric analysis o!' the carbon dioxide obtained indicated t,hat no water n-as pre.wnt in it, showing t h a t tematic error due to n-atcr carry-over. id present. T h e ultimate precision of tht, method depends on the reproducibility arid precision of the manometer measurement. This nil1 be of the order of 0.2% a t hcat. Fluctuations are noted, lio\vevcr, from day to da>-, c~orroI)oratii~g tlie d*ta of Saughton and Frotlyma ( 7 ) . These cannot tie : c . w r i k ~to any single i:ii*tor, surh as the ambient lat)oi,ator:. tempwature or atmospheric prwsure. The apparatus works 1x.t a t an ainliient temi? affected by alloiving perature of 23" to 25" C. Iiepi~oduc~il,ilit!-.surges of gas t o enter the t r a p section 1):. carele..;. manipulation of stopcock S2 (Figure l ) , undul>- estwiling the total time of the :inalysis, or not assuring that, the t r a p arc: nt room temperature at the time of pressure measurrmrnt. Thtsrinostating the \\-hole trap-volume-manometer section at a i'rn. degrees above i'oom trniperature would undoubtedly incwa,v the precision, hut this :idtled feature is not considercd nec For nitrogen-containing sul,stancw, g r w . ~ variations ivere found in conditions necessary t o ol)tain good results, and longer comi)ustions a t higher teniperatureR ivere required. The use of t h r copper gauze for reduction of t h r nitrogen oxides without riduring carbon dioxide to carbon monoside is in general satisfavtoi.y, hut the temperature at which the copper is held is a critiral factor. I t \vas found that from about 475' C. and up\\ :ii.ds, some carbon dioxide \ v a ~ tieing i~rcluctdh>-the mpper, as iiidicated in employing henzoic~acid in the nitrogen procedure. I3elo\l- 460" C., carbon values \ver.e high for S B S acetanilide, dioside pressure Ijy unindicating a rontribut,ion to the c~ai~l,on iwlucrd nitrogen osides. In none' oi' these e a ~ e swere the n-ater values seriously disturbed (Table I I . Typical results for various compounds appear in Table 11. ISOTOPIC ASSAY PROCEDURES
The methods for the isotopic assay of carbon dioxide and Ivater obtained by combustion of organic compounds are u-ell documented ( 4 4 , 9-12). Therefore, only t'hose methods are treated in detail which involve the handling of the carbon dioxide as such for carbon 13 assay, rarbon dioxide or barium carbonate for carbon 14 assay, and water for the Rubsequent deuterium or tritium assay.
Procedure for Carbon 13 Assay. ;Z series of collection tubes (labeled A in Figure 2 in the carbon dioxide transfer apparatus) ip required. These consist of a 12/30 T inner joint connected to a vacuum stopcock sealed t,o a closed section of borosilicate glass tubing 22 mm. in outside diameter and 10 cm. long. T h e volume is calibrated by xeighing in water at, a given temperature. Prior to a combustion in the train, one of the collection tubes is sealed to the carbon dioxide take-off joint shown in Figure 1 with Apiezon S grease. T h e tube is evacuated through stopcock 85, which is subsequently closed for the determination of the carbon dioxide pressure. JVhen this determination is completed, S5 is opened and tube A is immersed in liquid nitrogen, so that the carbon dioside distills into the tube. S.5 is closed after the desired amount of transfer has been made and the manometric pressure is noted if desired. The partially filled
ANALYTICAL CHEMISTRY
1302
collection tube can then be attached to the sampling manitoltl of a mass spectrometer for the assay. T h e carbon dioxide 0 ' ) tained in this TTay, if complete combustion is indicated by thv analytical values, is pure and representative enough for preciw W0r.k.
T h e heavy water t r a p can then be modified so t h a t a pipet can be inserted through an opened stoprock to remove the liquid water. This general procedure gives an assay material repI (-entative of original material as regards deuterium content.
Procedure for Deuterium Assay. The cornbustion train is modified by replacing the traiii bypass (Figure 1) by the h w v y water trap (Figure 2). T h e ilpiezon black wax is removed f i ~ ~ m the standard points by carbon tetrachloride and replaced h y Apiezon ?: grease. T h e heavy water trap is placed in this pwition in the train t o reduce t o a bare minimum the glass surf:tco.: available for the possible exchange of ordinary adsorbed w t e i . . T h e hydrogen analysis is sacrificed, b u t this is balanced I]>- th(1 fact t h a t the carbon analysis is still available to ensure, if correct. that a complete combustion has taken place. Several aliquots of the compound t o be analyzed and assayed are placed iii latinum combustion boats. plus one accurately weighed sample. &he boats and samples are kept i n a desiccator prior to coml)urtiox. For analysis an unweighed (usually qualitatively equivalent to the weighed amount) amount of the compound is burneci with the heavy water trap ininiereed in dry ice-acetolie and thcl c:irIion dioxide trap in liquid nitrogen. T h e first stopcock 011 the heavy water trap after thc, (,omhuetiont u l l e is then employerl in lieu of stopcock S2 for the i)leetting operation. .\fter the usual combustion and complete evacuation pror:etlure. t,lie carbon dioxide is pumped away, both stopcocke on the he:ivy miter trap are closed, and the bath is removed. T h e vaporized water is allowed to equilibrate Ivith what absorbed water i.; i i i the trap for 2 minutes. Then the vapor is pumped awa?. and the Figure 3. Filtration Apparatus whole procedure is duplicated with another unweighed portion of the sample. Finally the weighed sample is burned, the deuterated water collected. and the carbon percentage determined. T h e values obtained by t,liis procedure are shown in Tahle T h e heavy water trap can then he removed and affixed to :L 111. The phenyl (butj.1-2-di) ketone was prepared directly froin vacuum manifold as described b y Friedman and Irsa ( 5 ) . By the l-propanol-d2 and thus the deuterium atom per molecule distilling zinc diethyl directly into the heavy water trap immersc 1 in d r y ice-acetone i t is possible to eliminate exchange reactioii value should be t,he same. Other nonisotopic compounds were ot,lier than the direct reaction to produce deuterated rthane. liurned in t h e apparatus Iietveen the determinations on these which does not exhibit this trouhlesome exchange with adsorlieti t w o compounds. water and iT-hich can be tiirrct1)- employed for m i s s spc'ctroinrtricFor tritium determination the wat,er can be made to react with assay. CHJlgX t o produce CHaT for use in gas counting according to This method is not, suitable for the usual densit). deteriniliathe method of Robinson (9;. tions, as too small quantities of water are usually obtained. By Procedure for Carbon 14 Assay. COUNTIXGAS CARBOX and T T i , this can be overcome. adding a mu(-h larger ~ o l u n i eto DIOXIDE.For this procedure, the counting tuhes described by Uernstein and Ballentine ( 2 ) or Eidinoff ( 4 ) can be employed. In both cases, t,he first step is to condense the radioactive carbon Tahle 1. Optimum 'Teniperatttre f o r Reductor dioxide into the counting tube. T h e subsequent operations-of TD of CII Compuund C , c/o H, 5% sdding met,hane i n the first case or carbon disulfitlc i n thc. secontl 415 Benzoic acid 69.06 4.96 -and tlip circuits required for proper operation are indicated i n 461 Benzoic acid 68.69 4 09 the referenres. Still another alternative is to collect the (*arbon 475 Benzoic acid R5.89 4.96 670 Benzoic arid 60.65 4.99 dioxide in a previously ovacuated ionization chamber connected 460 Acetaniljde 70.94 6 72 475 Acetanilide 61 84 6 72 t o the take-off. The carbon dioxide need not he sublimed in, :IS 4-18 Acetanilide 72 64 6.72 the relative volumes usually are such t h a t an appreciable fraction Theory for benzoic acid 68 84 4.95 xi11 be in the chamber, which can be precisely measured. ThereTheory for acetanilide 71 08 8 71 fore, the only requirement of the counting tube (Figure 2) for Table IT. Specimen Analyses adaptation t o the combustion train is t h a t a 12/30 -$ joint is available, so t h a t t,he tube can be put on the carl)ori dioxidc take-off position.
n
Sucrose
42 10
Tetraphenyl porpliiii Dur.oi~uinone-irneth?I,-('I4
85.97 73.1
I*'uroir acid-(carhoxy)-C"'
53 58
Glycolic a c d C l 4
'
31 58
Clilorrmcetic acid
25 42
i l l e t l i ~ l -i-dinitrobenzoate ~, ~letIiyl-C'4-3..j-dinitrobenzoate
-12 49
Glyculic acid
42.83 41.63 41.90 86 30 73.28 1 2 08 72 74 53.73 53 61 31 80 31 36 25 65 24 90 42 83 42 q-1
6.48
6.72 6.46
4.92 7.37 3 60
5 30
3 19 2 67
6.38 4.96 7.40 7 41 7 40 3.50 3 56 5 27 5 40 3 00 3 08 2 64 2 70
Table 111. Analysis and .4ssay of Deuterium Compounds
c.
% Deuterium round Atoms/Molecule 58.17 1.75 57.83 1.78 Phenyl (butyl-2-dd ketone 80.43 80.64 1.78 81.58 1.67 0 Theory calculated on basis o i 2.0 deuteriuni a t o m per molecule 70 Ca. Theory 58.01
Cornpound Propanol-I-&
.
.~
I n routine work the counting tulle is evacuated through S5 and remains in position throughout t h r combustion, evacuation. and gas-measurement operations. ilfter the carbon dioxide is measured i t is distilled into the counting tube in1 nersed in liquid nitrogen by opening S5. .4s a complete transfer i? not necessary and would be tedious, about 955& is transferre 1 and S5 is closed. T h e residual amount in t h e gas measurement system is redetermined and this gives hy dieerenee the amount in t h e counting tube. By then closing the counting tube $topcock the tube can be removed to a vacuum manifold for tht. subsequent operations indicated. Figures 2, 3, and 4 shoLv COUNTING A S B.k~Inkf CARBONATE. the apparatus used. The solution of Ba(OH)*.BaC19used for prccipitation of barium carbonate is made as follows:
A saturated solution of barium hydroxide is made by adding a n excess t o freshly boiled water in a bottle protected by, ail Ascarite tube. T o 2.4 liters of t h e clear supernatant barium hydroxide solution in another nitrogen-filled bottle is added 480 ml. of a solution containing 51.6 grams of barium chloride dehydrate also made from freshly boiled water. This latter
V O L U M E 2 4 , NO. 8, A U G U S T 1 9 5 2
1303
solution is kept in a bottle protected with a tube of rlscarite and a siphon. For use, portions of the solution are run into a buret untiei, nitrogen. This solution is titrated against 0.1 S hydrochloric acid using phenolphthalein. T h e collection tube, B , has tiern desci,il)ed in the section on carbon 13 assay. A is connected by a standard joint and tubing 10 mni. in outside diameter, 8 cm. long, to a 2-mm. T-bore hollow-plug stopcock. One a r m of the stopcock has a 19/38 T outer joint and the other arm IS tynlarged to fit a vacuum hose. I n addition, a number of flasks consisting of 26-ni1. Erlenmeyer flasks, F , sealed to 19/38 T inner joints and corrwponding caps, C, are prepared (Figure 2 , insert a t upper left). The above equipment constitutes the precipitat ion apparatus. T h c filtration apparatus consists of two glass chimneys, CH-I anti C‘H-2 (Figure 3), made of tubing 19 mni. in outside diametc,r, 5.5 m i . long (for use with 2 sq. cm. frit), withshoulders for use with a stmilard 28/12 spherical joint clamp, and a porous glass frit. G F , anti frit holder, F-1 and F-2. The glass frits, as supplieti l)y (’oming, are fine porosity, 20 mni. in diameter, and not fire poliPhecl. The frits are held in a two-piece holder, machined ot stainlws steel (Figure 4 ) .
will give the amount of carbon dioxide absorbed, s h i c h is :in additional check on the manometric analysis. The neutral slurry can then he handled with relative impunity, since lit,tle or no carbon dioxide will be absorbed from the atmosphere. T h e slurry is poured into the assembled filtration apparatus and suction is gently applied. T h e barium carbonate settles in a uniform mat on the frit surface and is washed with freshly boiled distilled water, 1 to 1 methanol-water previously prepared, and finally pure methanol. T h e frit holder is rcmoved, the top surface of the holder F-1 is wiped o f f , and the unit is dried for 5 minutes at 115’ C., cooled, and stored in a microdesiccator. S o attempt, is made to filter o f f the barium carbonate quantitatively. Its weight on the frit can be readily tirtermined for calculation of the thickness for self-ahsorption corrections. An amount of hariuni carbonate is usually enipioyed equal to 130 to l5OYc of “infinite thickness.”
The advantage of this arrangemrnt i? that thc geoineti,y is cont,rolled by niachining of metal parts. Coneequrntly, the nrca of thc frit exposed can be ninde exwtly 1 sq. cni. (0.444 i 0.001 diameter) or 2 sq. cm. (0.628 i.0.001) and the surfare of t’he f r i t is alpvaye the same distance from the bottom of the holder, regardless of irregularities in the frits as to area or thickness. The dimensions of the holder shown in Figure 4 are such that the assembled frit, and holder can be used in all the commercially available flow-type counting equipment in addition to the usual geometricd setup for Geiger-Xluller tubes. X simple filt,ration is the onlj. manipulation required to ensure accurate and reproducible results. T h r prc~cipitatioriand filtration procedure is as foilon-r::
the reproducibility in the area was better than the reproducibility of samples running less than 90% of infinite thickness. Consequently, F i t h small amounts of barium carbonate, the use of a smaller area (1 sq. cm.) proved better than working at less than infinite thickness over a wider plate. Table IVYindicates the reproducihilit- obtained when portions of a homogeneous sodium carbonate solution were treated as indicated above and the resultant barium carbonate precipitates ivere counted on the 2 sq. cni. holders.
The collection tube, A , containing CIJOr is sealed to the transf‘ei, piece as shown in Figure 2. I n a volumetric flask, flushed with nitrogen, 4.00 nil. of barium hydroxide/bariuni chloride solution of a known t k e r is placed and the flask is put in position on thc transfer piece. The system is immediately evacuated through the T-stopcock with a water aspirator, care being esercised to swirl the solution to prevent bumping. \\.hen the pressure is about 20 mm., the T-stopcock is turned to disconnect the vacuum and connect A and the barium hydroxide fiwk. This flask is warmed in a ivatei, bath a t 80” C. and the tril)t, -1 :topcock is opened. Barium carbonate appears a t once, anti tht, tligestion and transfer are continued for 5 minutes. \\liile there is no need for a quantitative transfer, this can be accoinplished by cooling the alkaline solution carefully to liquid nitrogen temperature. The T-stopcock is turned to isolate the carl)on dioxide in flask F where the complete absorption occurs readily. If this is done, a further digestion is suggested. T o remove flask F containing the slurry, nitrogen gas is admitted at the vacuum arm of tho T-stopcock. The flask is removed hydroand the excess barium hydroxide is t,itrated with 0.1 c-hloric acid using phenolphthalein as a n indicator. This titer
The routine use of these thick samples has Jeveral advantages. S o adjustment’in the assa?. has to l)e made for samples less than 100% infinite thicknew S o special niariipulations are required to ensure uniformly thin c*ai,honatcsour(’es. It was found that
Table IV. Sample Holder N o . 2 7 17 3 22 11
Reproducibility MA. Yet
R ~ C O Weight, J 83 2 95 4 40.6 49.3 55 7 38 1
Coiinti ’11111
ACKNOW’LEDG.MENT
The authors wish to thank Italph \Vhite for his help i i i thc design and construction of the frit holders, Karl Walther for the r o n ~ t ~ ~ ~ coft i the o n various components of the combustion tr,airi, aiid G ~ o r g eCos for several of the tlrarvings. LITERATURE CITED (1) .%rmstrong, I\-.D., Singer, L.,
Zhai.sky. J. I%..arid Duiishre, U.,
S c i e n c e , 112, 531 (1950).
( 2 , Hernstein, \Y., a i d ~allciitiiie,I t . . Rer. Sci. I ~ L S / ~ W / L 21, ~IL/.S,
158 (1950).
(3) Calvin, 11 , Ileidelberner.
C., Reid, J . C , , Tolbert. B.AI.. and Yaiikwich, 1’. F.. “Isotopic Carbon,” S e w l o r k , p. 1 3 6 J o h n \Tiley 8sons, 1949. Eidinoff, XI. L.. ASAL. CHEM.,22, 529 (1950). Friedman, I>., and Irsa, A. P., Ihid., 2.1, 876 (1952j .
Kanien, 31. D., “Radioactive Tracers in 1%ology.” S e w York, Academic Press, 1948. Saughton, J. J . , and Frodynia. XI. >I,, AA-.~I,. C H E l f . . 22, i 1 1 (1950). Siederl, J . H., arid Siederl, V., “C)rgaiiii, (Juantitatire Xficwanalysis,” 11. 103, New I-ol,k, John \\?ley & suns, 1942.
Robinsoil, r.\.., Rei,. .Sei. -.078
I?islrilnierits, 22,
350
i1951).
Van Hlykr, 11. L)., Steeie, R.. a r i d Plazin, .J., J . B i d (,‘hcrr!., 192, 7 K l (1951). W-elzbarh, K. E., and Van Dyken, .L, Atomic. Energy (’oniniiasion, Documerit d E(’D 2998. JVilson. D. I$-..Sier. .\. 0. C., and Ruinanii. S. P.. “ P r e p r a t i o n and Measurement of‘ Isotopic Tracers,” .inn Arbor, Mich.. .J. \V.
Kdwards, 1946. 1-
.om Figure 4.
Dimensions of Frit Holder
R E ~ E I V Efor D review October 24, 19.51. Accepted .Tune I O , 1932. Researrli rarried o u t under the nuspicea of the U. P. Atounic Energy Cornminsion.
