1504
ANALYTICAL CHEMISTRY
recovery of carbonate ranged from 95 t o lOOyoin all compounds tested, with the exception of adenine which gave a value of about 80%. Persulfate combustion cannot be used for water-insoluble compounds and may be found unsatisfactory for the combustion of some stable ling compounds. Also unsatisfactory results TTere obtained in the combustion of methylamine, as observed also by Anthony and Long ( 2 ) . The results obtained n ith carbon-14-glucose and carbon-14succinate in a comparative study using the Van Slyke and Folch combustion fluid and persulfate can be seen in Table 11. The radioactive assay of these compounds by these tvio methods was found to be identical The flask uapd here can be used in general as a diffusion flask. Although its dimensions are not as efficient for diffusion as the conventional Conway dish (j), it has the advantage of easy evacuation, t h w hastening diffusion. These flasks are more convenient to use than Conw,y dishes, as they do not require greasing: and they are also much cheaper. Also, if required, reagents may be injected into the flask after it is closed. The persulfate oxidation can also be performed in a variety of vials or vewels adapted fox this purpose (3). Thus, using an ordinary Skrip ink bottle (the top well of iThich serves as an alkali well), recoveries of 95 t o lOOyowere obtained in the combustion of acetate, citrate, lactate, acetone, and glucose. The special value of such flasks is that they combine the characteristics of a reaction and diffusion vessel. Such flasks were used by the authors in a great variety of reactions that involve the formation of carbon dioxide, such as the decarboxylation of lactate, amino acid assay with ninhydrin, and the complete degradation of carbon-14-acetate. ACKNOWLEDGMENT
The authors wish to acknodedge the gifts of succinic acid-2carbon-14 from B. M.Tolbert and of recrystallized adenine and thymin from Carl Emanuel. LITERATURE CITED
(1) Abraham, S., Putman, E. W., and Hassid, W. Z., Arch. Biochem. Bzophys., 41, 61 (1952).
Table 11. Comparison of Van Sly-ke and Persulfate Combustion of Carbon-14 Compounds Glucose was prepared photosynthetically ( 9 ) and was shown t o be erenly labeled ( I ) . Succinic acid used was labeled with carbon-14 in the 2-position ( 7 ) . Identical aliquots were combusted in each case. The Van Slyke combustion was carried out according t o Barker (4)and all samples after the addition of barium chloride and titration of the excess alkali were mounted on filter paper according t o Entenrnan e f al. (6) and assayed for radioactivity on a conventional end-window counter. Persulfate Oxidation Barium Specific carbonate, activity, mg. c./min./mg.
Van Slyke Oxidation Barium Specific carbonate, actlvlty, mg. c./min./rng.
Glucosecarbon-14
35.1 37.3
9.1 9.4
37.5 36.5
9.2 9.3
Succinic acid-2carbon-14
18.2 16.4
21.1 20.5
18 3 18.0
21.2 20.7
Blank
0.5
0.8
Anthony, D. S., and Long, 31. V., Oak Ridge Xatl. Lab., Rept. 1303 (1052). CHBM.,26, 1504 Baker, N., Feinberg, H., and Hill, R., ;ISAL. (1964). Barker, H. A . , in “Isotopic Carbon,“ by Calvin AI., Heidelberger, C., Reid, J. C., Tolbert, B. AI., and Yankwich, P. E., p. 93, New York, John Wiley &- Sons, 1949. Conway, E. J., “MicrodiffusionAnalysis and Volumetric Error,” 1st ed., Sew York, D. Van Kostrand Co., 1940. Entenman, C., Lerner, S. R., Chaikoff, I. L., and Dauben, W. G., Proc. SOC.Esptl. Bzol. M e d . , 70, 364 (1949). Jorgenson, E. C., Bassham, J. .i.,Calvin, A f . , and Tolbert, B. AI., J . Am. Chem. SOC.,74, 2418 (1952). Osburn, 0. L., and Werkman, L. H., ISD. ENG.CHEM.,; I N ~ L . ED.,4, 4421 (1932). Putman, E. W.,and Hassid, IT. Z., J . Biol. Chem., 196, 749 (1952). Thorn, J. A , , and Shu, P., C a n . J . Chem., 29, 558 (1952). Van Slyke, D. D., and Folch, J., J . Bzd. Chem., 136, 509 (1940). Weinhouse, S., in “Isotopic Carbon,” by Calvin, M.,Heidelberger, C., Reid, J. C., Tolbert, B. AI., and Yankwich, P. E., p. 94, Kew York, John Wiley &- Sons, 1949. RECEIVED for review July 6, 1953. Accepted April 5 , 1954.
Analytical Procedures Using A Cornbined Cornbustion-Diffusion Vessel Simple Wet-Combustion Method Suitable for Routine Carbon-1 4 Analyses NOME BAKER, HAROLD FEINBERG, and ROBERT HILL Radioisotope Unit, Veterans Administration Hospital, Cleveland, Ohio, and the Division of Physiology of the University o f California, School o f Medicine, Berkeley, Calif.
R
ECEKTLY Michaels et al. ( 4 ) described the use of a screwcap bottle for the quantitative oxidation of p-hydroxybutyrate to acetone. By using a screw-cap bottle which has a smaller compartment within, the authors have recovered carbon dioxide from the semiquantitative oxidation of a series of organic compounds with Van Slyke-Folch combustion fluid ( 5 ) . The method, though less quantitative than the original Van SlykeFolch procedure ( 5 ) ,has proved satisfactory for the routine assay of isotopically labeled, dry, nonvolatile compounds. APPARATUS AND REAGENTS
Screw-cap bottles of 0.5- to 2-ounce capacity with a glass well, either permanentlv attached (Figure 1, A ) or removable (Figure f,, B ) . Sheaffei’s Skrip writing fluid bottles “with the top well, 2-ounce capacity, were used successfully as combustion vessels. The enclosed well need not be in a central position for quantitative diffusion-absorption of carbon dioxide into sodium hydroxide. Bottle caps, either metal or plastic, equipped with a full rubber
gasket (Goodrich Ameripol-D rubber, ‘/IB inch thick) previously washed in boiling soap solution. It is suggested that an extra supply of caps be kept available to replace the metal caps, which rust readily, or the lastic caps, which may crack when tightened. Carbon dioxidegee sodium hydroxide, 31V. Van Slyke-Folch combustion mixture ( 5 ) . PROCEDURE
Pretreatment of Apparatus. The recommended treatment of the apparatus prior to its use is similar to that commonly employed in wet-combustion procedures ( 2 , 5 , 6). Bottles and wells are heated in chromic acid cleaning solution, rinsed, and ovendried. About 5 ml. of the combustion mixture are then added to each bottle; the system is capped and heated for 30 minutes at 15 pounds pressure in either an autoclave or pressure cooker. If not treated in the above manner, new rubber gaskets, even though previously washed in boiling soap solution, may produce 8 to 9 mg. of barium carbonate blank values. After an apparatus has been used for an oxidation, it is rinsed with distilled water, dried a t room temperature, and stored inverted in a drawer. If the bottles and wells are not pretreated with hot
1505
V O L U M E 2 6 , N O . 9, S E P T E M B E R 1 9 5 4 Table I. Recovery of Barium Carbonate Following Van Slyke-Folch Wet Combustion of Organic Compounds i n Screw-Cap Bottle Combustion Fluid, MI.
Compound Oxidizeda Fructose
5
Theoretical Carbon Dioxide Barium Carbonate "'g. Recorered, ' 7 0 Theoretical Recovered 76.3 75.2 98 94 70.0 65.8 122 126
121 126
99 100
Potassium oxalate
5
Leucine
J
66.7 54.2
65.8 54.5
99 101
Cholesterol
J
90.8 99.3
84.2 89.2
93 90
Benzoic acid
3
238 118 97.0
218 110 95.5
92 93 98
Glucose
3
59.2 109 115
56.2 102 114
95 94 99
Phenylglucosazone
3
133 120 109
121 107 92.0
91 89
3
141 140 110
119 118 89.5
84 84 81
10
119 173
110 160
92 93
Cholesterol
Phenylglucosazone Average blank Blank (sodium hydroxide alone wresent during combustion period) Blank [carbon dioxide regenerated (S)] a
85
RESULTS
Tables I and I1 list th: recoveries of carbon dioxide following the oxidation of a variety of compounds by the above procedure. The proper blank value was subtracted in determining each yield.
Table 11. Recovery of Carbon-14 Follow-ing Van SlykeFolch Wet Combustion of Organic Compounds in ScrewCap Bottle Compound Oxidized F a t t y acidcarbon-14
+ 0.7
3 t o 10
0
0
0
2.7
...
3
0
3.0
...
4.7
bustion mixture acts as a desiccant, the contents of the absorption well may become completely dehydrated: nevertheless, the sodium carbonate-sodium hydroxide can be recovered completely by redissolving the material in water. A thin white film of material may remain in the absorption well if it dries out completely, even after five water washes. However, this precipitate represents a negligible amount of carbonate in these experiments. The carbon dioxide recovery was determined by adding an excess of barium chloride to the sodium carbonatesodium hydroxide solution to precipitate the barium carbonate. The excess base was titrated to the phenolphthalein end point. Bromocresol green was then added and the barium carbonate n-as titrated to the bromocresol green end point with standard dilute hydrochloric acid.
Analytical reagent grade compounds not dried before weighing.
Glucosecarbon-I4
A4ctivity Addeda' 21.8
Activity Recovered' 22 7 21.0 21.7
% of Added
Carbon-14c Recovered 105 98 101
30.0
31.2 104 28 5 95 103 30.8 Phenylglucosazone6.06 6.06 100 carbon-14 5.23 4 77 91 3.93 3.69 94 a F a t t y acid-carbon-14 and phenylglucosazone-carbon-14 assayed by method of Barker ( I ) . Glucose-carbon-14 determined by persulfate oxidation ( 8 ) . Activity a s counts per second, except for phenylglucosazone. The latter activitv is exwressed as counts wer second wer niiilisram of ducose carbon. I n case of phenylglucosazone-carbon-14, "per cent of theoretical specific activity."
*
RUBBER QASKET Cot- FREE NOW
METAL CAP
@
+
SAMPLE CCUBUSTION MIXTURE
RUBBERQASKET METAL CAP COP- FREE NOOH
@
SAMPLE COMBUSTION MIXTUR
Figure 1. Screw-Cap Bottles Used as Combustion Diffusion Vessels cleaning solution, rinsed with distilled water, and oven-dried before use, a larger and more variable blank value (8 to 12 mg. of barium carbonate) Fill result. If special care in storage is observed, one should be able to carry out oxidations in the bottles without further treatment (6). Combustion and Removal of Sodium Hydroxide-Sodium Carbonate. An aliquot of organic material is placed in the bottle and the solution evaporated to dryness. Into the well 1 ml. of carbon dioxide-free sodium hydroxide is delivered. Approximately 5 ml. of the combustion mixture are then added to the main compartment. The cap is immediately screxed securely in place. A rough surface strip of rubber has been found useful as an aid in tightening the caps. The bottles are then placed in a pressure cooker or autoclave a t 15 pounds' pressure (250' F.) for approximately 30 minutes, a t which time the sodium carbonate-sodium hydroxide may be quantitatively transferred into a suitable container by aspirating the alkaline carbonate with a syringe equipped with a blunt needle. It is usually convenient to allow the bottles to cool before removal of the well contents; however, all of the carbon dioxide has been absorbed by the base during the 30-minute heating period. Since the com-
The authors have been unable to reduce the blank below 4 mg. of barium carbonate by merely cleaning the apparatus according to the procedure described earlier. Regeneration of the carbonate a t room tempeiature in a flask such as has been used by Kats et al. (S), lowered the blank approximately 2 mg. of barium carbonate below the 5 mg. value which has been found in most of these experiments. From 2.5 to 3 0 mg. of barium carbonate were found after subjecting 1 ml. of 3 5 sodium hydroxide to the high temperature and high pressure conditions of the pressure cooker in the absence of combustion mixture. This value is two to three times that obtained under the less severe conditions of the persulfate oxidation (3). The blank value does not seem to vary either with the amount of combustion mixture used or with the size of the rubber gasket (Figure 1,A and B ) . By covering the synthetic rubber with aluminum foil, the authors were unable to reduce the blank below that found when only the rubber gasket was used. However, despite the rather large value of the blank material, the error introduced by this variable need not be serious. In determining 16 such values on three separate occasions only three samples deviated by more than 0.5 mg. of barium carbonate from an average value oi 4.7 mg. By using sufficiently large aliquots of carbon to yield 0.5 to I m M carbon dioxide, the error introduced by variation in the blank should rarely exceed 1%. I n assaying specifically labeled compounds it is important to establish that all of the carbon atoms hava been oxidized to the same degree. Since 100% yields have not been obtained consistently in the present method, before using this method its validity should be determined by comparison with an established
ANALYTICAL CHEMISTRY
1506 method for the complete oxidation of the compound to carbon dioxide (6, 6). For example, after oxidizing, according to the authors' method, phenylglucosazone-carbon-14 labeled in the glucose portion of the molecule, the authors found that the specific activity of the glucose carbon averaged 95% of that obtained by direct plating of the osazone on paper (Table 11),even though in some cases only 90% of the theoretical yield of carbon was obtained (Table I ) . This might be taken to indicate that preferential oxidation of carbon atoms may not be a major source of error here. However, since evenly labeled glucose was used in preparing the osazone, it is possible to obtain such a high recovery of the theoretical specific activity even if one or more of the glucose carbons were only partially oxidized to carbon dioxide. Hence, before using the method for the determination of specifically labeled glucose, the suitability of this oxidation technique should first be established.
ACKNOWLEDGMENT
The authors gratefully acknowledge the technical assistance of C. L. Hannum, Jr. LITERATURE CITED (1) Barker, H. A., in Calvin, If.,Heidelberger, C., Reid, J. C., Tolbert, B. & and 'I. Yankwick, , P. E., "Isotopic Carbon," p. 93, New York, John Wiley & Sons, 1949.
(2) Claycomb, C. K., Hutchens, T. T., Van Bruggen, J. T., and Cathey, W. J., Nucleonics, 7, 38 (1950). (3) Katz, J., Abraham, S.,and Baker, K.,Aa.4~.CHEM.,26, 1503
(1954). Michaels, G. D., Margen, S., Liebert, G., and Kinsell, L. W., J . Clin. Invest., 30, 1483 (1951). ( 5 ) Van Slyke, D. D., and Folch, J., J . BioZ. Chem., 136, 509 (1940). (6) Van Slyke, D. D., Gteele, R., and Plazin, J., I b i d . , 192, 769 (1951). (4)
RECEIVED
for review July 9, 1953.
Bccepted March 29, 1954.
Thermolysis of Zinc Monosalicy laldoxime JOSEPH RYNASIEWICZ
JOHN F. FLAGG
and
General Electric Co., Knolls Atomic Power Laboratory, Scbenectady, N. Y.
P
EARSON ( 4 ) attempted to precipitate zinc as zinc disalicylaldoxime, Zn(C,H602X)2,from solutions of pH 7 t o 8, but
the result was a salt of variable composition. On the other hand, Flagg and Furman ( 3 ) found that if such a precipitate were digested for 10 minutes between 90" and 100" C., zinc monosalicylaldoxime, Zn( CrH,OzN), was formed. The compound came to constant weight when dried a t 110" C. for 1 hour and remained stable for several hours thereafter a t the same temperature. The dry zinc salt contained 32.5'% of zinc compared with the theoretical 32.6% of zinc found in Zn(C7H602S). Furthermore, this compound was used successfully as a weighing form for the analysis of zinc in a National Bureau of Standards brass sample. Duval (1) and coworkers obtained thermolysis curves for a zinc complex which they believed was Zn(C7H502iY), but they were unable to determine a satisfactory drying temperature for the compound. As a result they proposed that the method be abandoned.
The correct drying temperature for zinc monosalicylaldoxime lies between 25' and 285" C., depending on the moisture content of the sample and the heating (drying) rate. Practically, drying to constant weight a t 110' C. is recommended. As observed by Duval ( I ) , the wet sample starts to lose moisture a t -60" C., and decomposes very rapidly a t 290" C.
Table I.
Curve, Fig. 1
I 11
I11 IV
b d e
OC
I
0
1
100
ZW
Dc,
,
100
I
I
100
700
900
I
1000
Figure 1
Reagent Added for P p t n . of Zn Excess Deficiency Deficiency Deficiency Excess
a
Thermolysis" of Zinc Salicylaldoxime hfoisture in P p t . a t S t a r t of Thermolysis, % None
Discontinued Heating for 1 %ur at C.
Temp. Range for Zn(X),
Zn in Zn(X)b,
110
-5Od
115
25-285 115-240 135-290 2 15-29 0
32.0 43.0 37.4 40.5
245-315
33.0
-5Od 250e 63OC
135
Heating uninterrupted Heating uninterrupted
c.
%
Heating rate 5' C. per min. Zinc in Zn(CrHa0zX) is 3 2 . 6 % . Air-dried for -70 hours a t 25' C . Air-dried for -24 hours a t 25' C. Wet precipitate immediately after filtration
A zinc complex of variable composition (probably a mixture of zinc hydroxide and zinc salicylaldoxime) resulted when less than the stoichiometric amount of salicylaldoxime reagent was used for the precipitation of zinc. When a 20% excess of reagent was used, zinc monosalicylaldoxime, Zn(C7HsO*N), was formed. In all cases, a constant weight for the final ignition residue w ~ t s obtained between 500' and 1000' C. This observation is a t variance with the Duval's thermolysis data ( I ) , which showed that the zinc salicylaldoxime complex is converted to zinc oxide a t 950' C. LITERATURE CITED
(1) Duval, C., "Inorganic Thermogravimetric Analysis." p. 283,
Kew York, Elsevier Publishing Co., 1953. Using the same type Chevernard thermobalance employed by Duval (1, 3), this laboratory obtained thermolysis curves which confirmed the drying temperature and the composition of the zinc salicylaldoxime complex proposed by Flagg and Furman (3). The data for five thermolysis curves (Figure 1) are summarized in Table I. The following conclusions were made.
(2) Duval, C., et nZ., A N ~ LCHEM., . 23, 1271-85 (1951). (3) Flagg, J. F., and Furman, H. N , IWD.ENG.CHEM.,ANAL. ED.,
12, 663-5 (1940). (4) Pearson, Th. G., 2. anal. Chem., 112, I79 (1938). RECEIVED for review J a n u a r y 15, 1954 Accepted M a y 24, 1954 The Knolls Atomic Power Laboratory is operated b y the General Electric Co for t h e Atomic Energy Commission T h e work reported here was carried o u t under Contract TV-31-109 Eng-52
