1482
ANALYTICAL CHEMISTRY
down to the level to which the mixture has run. T o eliminate disturbances of the surface when adding solution to the column, a special device used. This consisted of a funnel with the stem end cut off at right angles to the stem and with a flat piece of glass, inch in diameter, fused across t'he end by means of three g l a s bead?. This provided a baffle t o divert the descending fluid into horizontal flou-. In determining the proper conditions for packing columns and for adding mat,erial and solvent, a visible check was provided by using a dye, Calco Oil Blue N.A., with 2.5% isopropyl ether in hexane as the developing solvent (Figure 2). -432 X 300 nun. column contains 125 grams of adsorbent and requires approximately 1 hour to pack by the slurry method. Although parking bj- this method is slower than by tamping the dry adsorl)ent, the greatly improved evenness of zones justifies the additional time in packing.
Hagdahl, L., Acta Chem. Scand., 2 , 574 (1948). Holmes, H . N . , Cassidy, H., AManly,R. S., and Hartzler, E. R., J . Am. Chern. Soc., 57, 1993 (1935).
Hurd, C. D., Thomas, G. R., and Frost, A. A , , I b i d . , 72, 3733 (1950).
James, A. T., Martin, A. J. P., and Randall, S.S..Biochem. J . , 49, 293 (1951).
Jeffrey, R. K., ASAL. CHEX, 23, 936 (1951). Kirchner, J. G., Mller, .I. M.,and Kelier, G. J., Ibid., 23, 490 (1951).
Levy, A. L., Chemistry & Industry, 1945, 380. Partridge, S. XI,,Biochem. J . , 44, 521 (1949). Schwab, G. hl., and Jockers, K., d n y e w . Chem., 50, 546 (193i). Tiselius, A . , A r k . Kerni iMineral. Geol., 14B, No. 22, 1 (1940). Ibid., No. 32, 1 (1940). Tiselius, A., . l ' a t u r ~ i s s e n s c h ~ n37, , 25 (1950).
Tiselius, A , , "The Svedburg 1884-1944," p. 370, Uppsala, illmqvist and Wiksells, 1944. Tiselim, A., and Claesson, S., A r k . Kenii Mineral. GeoZ., 15B, h-0. 18, 1 (1942).
Whistler, R. L., and Durso, D. F., J . A m . Chem. SOC.,72, 677 REFERENCES
(1950).
Rei,oza, >I., ASAL. CHEM.,22, 1507 (1950). Cassidy, H. G., J . Am. Chem. Soc., 63, 2735 (1941). Castle, D. C., Gillam, A. E., Heilbron, I. M., and Thompson,
H. XT-., Biocheni. J . , 28, 1702 (1934). Claesson, S., A r k . Kemi Mineral. Geol., 23A, N o . 1, 1 (1946). Cleaver, C. S., Hardy, R. 9., Jr., and Cassidy, H. G., J . Am. C'hmn. Soc., 67, 1343 (1945).
Euler, H., and Schlenk, F., 2. physiol. Chem., 246, 64 (1937). Glcnn, R. .I.,Wolfatth, J. S., and DeU'alt, C. W., Jr., ANAL. ('HEM.,
24, 1138 (1952).
Winterstein, d.,and Stein, G., 2. physiol. Chem., 220, 247 (1933).
Zechmeister, L., and Cholnoky, L., "Principles and Practice of Chromatography." p. 67, New York, John Riley & Sons, 1941. RECEIVED for review iMarch 4. 1952. Accepted April 2 6 , 1952. Report of a s t u d y made under t h e Research a n d Marketing Act of 1946. T h e mention of special instruments or materials throughout this paper does not imply t h a t they are endorsed or recommended b y t h e Department of Agriculture over others of a similar nature not mentioned.
Radiocarbon Combustion and Mounting Techniques ERSEL A . EVANS
AND J. L. HUSTON Oregon State College, Corvallis, Ore.
THE course of H-ork on exchange reactions in the solvents 1Sacetic acid and acetic anhydride ( 4 ) , and on a n isotope effect
on the rate of combustion of sodium acetate by chromic acid (3). useful modifications were developed of published procedures for performing wet combustions t o give barium carbonate, and for mounting barium carbonate in very small amount for radioassay. In preliminary work on wet combustions, much difficulty was encountered with blanks due to carbon dioxide from the air and t o sulfur trioxide evolved from the Van Slyke-Folch oxidizing mixture, the latter difficulty being especially troublesome because sodium acetate was difficult to oxidize and required considerable heating of the oxidizing fluid. Figure 1 showe the apparatus used t,o eliminat'e these blanks. The design of this apparatus was based largely on t h a t described by Lindenbaum et al. ( 6 ) , but differed from theirs in permitting more convenient introduction of fikered barium hydroxide solution and in dispensing with a bubbling tube, carbon dioxide being absorbed in vacuo. It !vas also designed to permit positive elimination of barium sulfate blanks by redistillation of carbon dioxide. The authors believe the use of barium hydroxide solution is more convenient than the use of carbonat'e-free sodium hydroxide, b u t the use of the latter reagent m-ould be preferable if it were desired to determine the amount of carbon dioxide evolved as well as t o make a sample for radioassay ( 2 ) . Combustion took place in the lo\yer flask, C, the stopcock, S3, and ground joint of which were lubricated with a mixture of phosphorus pentoxide and phosphoric acid; all other ground surfaces were lubricated xvith stopcock grease. The sample (usually about 50 mg.) was placed in this flask, the apparatus was assembled and evacuated t,hrough stopcock S2 by means of a n aspirator, barium hydroxide solution was admitted from one of t h r upper cups, J , through its sintered-glass filter, and 82 was closed. The c,ombustion fluid was then admit,ted to the lower flask by means of S3. S o potassium iodate was used ( 2 ) . The large stopcock, F , could be used to allow the combustion flask to communicate with either side; the apparatus was designed t o permit taking two barium carbonate samples when
the isotope rate effect was being studied, and could be simplified when used for simple routine combustions alone. The combustion flask was heated with a small flame for 10 to 15 minutes (a much shorter period would be required if a substance more readily oxidizable than sodium acetate were being used), and was allowed to stand another 15 minutes, the barium hydroxide solution being stirred all the while. The stirring was done by means of a small glass-enclosed bar magnet, actuated by a Precision Scientific magnetic stirrer. Barium carbonate free of barium sulfate was now prepared as follows. P was turned to close off the barium hydroxide from the combustion flask, air was admitted, and this flask was removed and replaced by another flask which was exactly alike except that the side cup contained a sintered-glass filter. After re-evacuation, barium hydroxide solution was filtered into this flask, hydrochloric acid was run in from the upper cup, J , and the evolved carbon dioxide was absorbed as before. When the absorption was complete the lower flask wvns removed and its contents were filtered quickly. Rather than follow the normal procedure of preparing Van Slyke oxidizing fluid by heating a mixture of fuming sulfuric acid, sirupy phosphoric acid, and chroniium trioxide ( 7 ) to 150" C. to accomplish solution of the chroFigure 1. Wet Combustion mium trioxide, oxiApparatus dize organic mate-
V O L U M E 24, NO. 9, S E P T E M B E R 1 9 5 2
1483
E a
C
140
I
Figure 2. C e n t r i f u g a t i o n M o u n t i n g Apparatus
L
rial, and expel sulfur trioxide, the authors preferred to heat the two acids together to 160" t o 180" C. until sulfur trioxide evolution ceased and then, after cooling to 140' to 150 to add chromic anhydride in a finely powdered form. A shorf period of shaking served to dissolve the chromic anhydride without further heating. In this x a y the evolution of any substantial amount of sulfur trioxide during combustions was prevented, and the osidizing power of the solution was not, destroyed during its preparation. &% solution prepared in this manner has been found satisfactory for use after standing 2 months, while solutions prepared in the manner first described must be discarded every week or two. The recommendation of Linderbaum et al. ( 6 ) , that the three substances be brought to 160" C., and held a t 140" to 160' C. for 15 minutes, produces very marked decomposition. Hutchens et al. ( 6 ) have reported a high-precision method of preparing barium carbonate samples for radioassay by centrifugat,ion. The procedure, which takes considerable time, was for preparation of infinite thickness samples, Tvhich naturally required sizable quantities of barium carbonate. JVe have found t,hat the apparatus shown in Figure 2 could be used to mount fairly small samples for radioassay m-ith a precision of about 2%, provided the samples xere well ground beforehand; preparation by the usual method of evaporation gave, when only a fely milligrams could tie mounted, samples much less uniformly deposited and consequently subject to larger errors in radioassal-. In this device, the brass cap, D , was machined to fit the Teflon ring, F , tightly and to hold i t against the Tracerlab copper planchet, .I. The cylinder, E , was made to fit into a standard centrifuge cup holder a t t h e top (catalog Xo. 325, International clinical centrifuge), and was machined as shos-n at, the bottom to fit snugly against a planchet a t R. Any slipping of t,he planchet xhile the cap was screwed on or off vias prevented by a emall
ridge, C; this avoided the customarj- use of an inner sleeve (1, 5 ) with its consequent increase in weight. I n use, the device \\as assembled as indicated, warmed slightly under a heat lamp, a few drops of a dilut,e suspension of barium carbonate were placed on the planchet and, then the device was centrifuged in a desk-top model centrifuge until the etheralcohol mixture had evaporated from the carbonate deposit. The time required depended upon the amount. of the suspension used. If small amounts Tvere used the evaporation was usually complete within a few minutes. The accuracy of the process was greatest if the amount of ether-alcohol used to make the suspension as kept as small as possible. Since these thin samples n-ere prepared on copper planchcts which \rere usually faintly visible through the barium carbonate deposits, it n-as possible to verify the uniformity of the deposits visually, as yell as by uniformity of rounting data. The follo\ving data of typical uniformity 11ere obtained from a uniform \jatc,h of bariuni cartionate. Satiii,le .4
Bariiiui Carboriare AIorinted. AIg.
Specific .\rti \-it? (Corrected fur. Self-Ahiorlitiiin)
14,11 1 4 90 5.60
80 i 82 4 81 3
B
c
The self-absorption rorrections rvere made with a c*urve prepared from heavier samples mounted by evaporation. Since it m-as found that, the corrected specific activities of thin niountings (prepared by centrifugation) agreed n-ell with thicker mountings (prepared by heat-lamp evaporation) from the same tiarium carbonate, it follows that self-al)sorpt>ion c,haracteri*tics are essentially the Pame in both cases. LITERATURE CITED
(1) Calvin. AI., Heidelberger, C., Reid, J. C..Talheit. 13. F,, and Tankwich, P. E. "Isotopic Carbon," Chap. 7 , S e w Tork, .John Wiley 8; Sons, 1949. ( 2 ) Claycomb, C . K., Hutchelis, T. T..and Yaii Bruggcn, .J. T., Snclromz'cs, 7, No. 3, 38-41 (1950). (3) Evans, E. -4.. and Huston, .J. L.. ,I. C ' h ~ m P . h ~ s . 19, , 1214 (1951). (4) Evans, E. .L, Huston. J. L.. and Sowis. T. H.. J . -im. Chern. Soc., to lie published. (5) Hutrheiis, T. T., Claycomb, C. IC., Cnthey, \T. J . , and \.an Bruggc11, J. T., Sucleuiaics. 7, S o . 3, 41-4 (1950). (ti) Lindenbaum, A , Schubei,t, ,J., and ;Ir.mstrong, IT, D., .\S.\L. C H E X . , 20, 1120 (1948). i i ) Van Hlyke, D. D.. and Folch, J . , .I. B i d . L'hem., 136, 513 flH40). H E C E I \ E Vf o r review J a n u a r y 28, 1032. Accepted AIay 9, 1932. I'uhlished with the approval of t h e Orerou S t a t e College AIonographs Cotrimittee, Researrh Paper S o . 202. Ilepartiiient of Cheiniatry, School c j f $rielire. W-orli was supported by a fellowshil> granted E. A . E v a m b y E. I . du I'ont de S e t n o u r s 8- Co., Inr.. aud by c o n t i a r t . i T ( 4 , j - l ) - 2 4 4 bet\reen I-, S. .itoniic Eneigy Commission a n d Oregon S t a t e Cullere.
Infrared Spectrum of Deuterated Ascorbic Acid Note on Labile Hydrogens JOHN W. WEIGL' G-nicersity of Minnesota, Minneapolis, Minn. S T H E course ot' research on a reaction photosensitized by
chlorophyll ( 1 7 ) the author prepared some deuterated ascorbic acid by exchange v-ith heavy x-at'er. An infrared spectrum n-as run in order to ascert,ain that deuteration had taken place; it turned out t,o be sufficiently interesting t o warrant more careful esamination. For purposes of comparison the xriter was fortunate to obtain, in addition, samples and spectra of tm-0 derivatives of ascorbic acid, in which two and four hydroxyls, resprctively, had been removed (10). The results were considered worth publishing for three reasons. (loniparkon of the spectra of normal ascorbic acid, deuterated 1
Present address. Ohio S t a t e UniT.ersity, Columbus, Ohio,
ascorbic acid, and the two derivatives. permitted the identification of a considerable number of their absorption bands. The spectra revealed that in ascorbic acid a t least one of the hydrogens attached to carbon is readily exchangeable, although these are often considered to be nonlabile. These observations also demonstrate the power of the conibined use of the mass spectrometer and the infrared spectrometer in the analpi!: for deuterium in organic compounds: The former provides a quantitative measure of the over-all tracer content, xhile the latter shows the distribution of isotopes among the various hydrogen positions in the molecule. EXPERI\IENTAL
Deuteration of Ascorbic Acid. P.S.P. ascorbic acid, 6.158 grams (35.0 mmoles), n-as dissolved in 30.0 grams of heavy
