Analytical Procedures Using a Combined Combustion-Diffusion Vessel An Improved Method for the Degradation of Carbon-1 4-labeled lactate and Acetate JOSEPH KATZ, S. ABRAHAM, and 1. L. CHAIKOFF Department of Physiology, University of California School of Medicine, Berkeley, Calif.
capped and evacuated through a hypodermic needle ( 6 ) . The vessels were heated for 30 minutes in an oven set a t 80" C., and allowed to cool. The barium carbonate was precipitated and assayed in the usual manner (6). The acetate was recovered by steam distillation of the permanganate solution in a Markham still ( 7 ) ; 20 volumes of distillate were collected, and t h e acetic acid was titrated x i t h alkali. Results of typical experiments with variously labeled lactates are presented in Table I. Degradation of Acetate (Equation 2). The sodium acetate solution was evaporated to dryness on a steam bath, and the H E use of combustion-diffusion vessels of very simple conresidue was taken up in a small volume of water (exactly 1 or 2 m1.). Aliquots were taken for Persulfate combustion ( 6 ) and for struction in analytical procedures has been described ( 2 , 6). decarboxylation by the Schmidt reaction. The carbon dioxide T h e application of t,his technique to the degradation of carbon-14obtained from the persulfate combustion is derived from the CY labeled lactate and short-chain fatty acids is reported here. and p carbons of lactate. Although several methods for the degradation of these comThe aliquot w e d for t h e Schmidt reaction was transferred to a shell vial and evaporated to dryness. About 0.2 ml. of 100% pounds are available (1, 3, 8, 10,I d ) , they do not lend themSdfUriC acid ( 8 ) was added, f o h w e d by the addition of about selves readily to simu~taneousdeterminations, and often require 30 mg. of recrystallized sodium azide. The contents were mixed specialized and expensive equipment. with a short stirring rod which was left in the vial. The procedure used here involves the oxidation of lactate to The vial was put in t h e renter well of the reaction flask, and t h e flask was capped and evacuated. I n most cases the Schmidt acetate and the degradation of the latter by means of a modificareaction does not begin until t,he flask is heated, but with some The degradation scheme is tion of the Phares method samples of 1 0 0 ~ 0sulfuric acid, considerable heat was evolved outlined below. immediately after t,he addition of the acid to the acetate-azide mixture. For this reason, an alternate procedure was employed. Degradation Scheme The vial containing acetate and azide was placed in the center CHsCHOH-COOH KMn04, H + CHrCOOH COz ( 1 ) well of t h e flask, and t h e flask was capped and evacuated. T h e (3) ( 2 ) (1) acid was cautiously injected into the vial v i t h 0.5-ml. syringe t,o (3) (2) (1) which was attached a %inch, 23-gage needle. CHa-COOH NaNs, H2SOcCH&Hz COZ (2) The flask was placed in an oven, a t about 80" for an hour. (3) (21 ' (3) (2) After cooling, about 2 ml. of carbon dioside-free alkali were injected into the main compartment of the flask with a hypodermic CHsSH2 KMn04, OH- COZ (3) syringe. One hour was allowed for carbon dioxide absorption, (3) (3) and the vacuum was then released by inserting a hypodermic needle through t h e cap. The vial was removed for subsequent EXPERIMENTAL methylamine distillation. I n the Schmidt reaction, t h e sulfur dioxide formed is also absorbed by the alkali. T~ eliminate this it was The combustion-diffusion vessel has been described (6). It necessary to regenerate the carbon dioxide. Base was delivered ~ ~ ~ $ ~ ~~ ~ f k ~~ ~ ~. ~ t h~e center d ~ ~ the t flask ~ was ~ capped h r evacuated. ~ ~ ~ into well, and and cap. However, a Of vessck Or screw-top vials can The carbon dioxide was liberated by injecting several milliliters be employed ( 2 ) . of 2'V sulfuric acid containing 1 to 270 of hydrogen peroxide into Oxidation of Lactate (Equation 1). Dichromate has been t h e main compartment. The hydrogen peroxide served to oxiused for decarboxylation of lactate to acetate ( 1 2 ) . In the dize the sulfite, and eliminated the variable blanks obtained authors' hands, however, about 10% of the a-carbon was also Use otherwise. The carbon dioxide was collected and assayed as oxidized, a finding in agreement with that of Daus et al. described previously' of permanganate as oxidant reduces contaminat>ionto 2 to 3%. The amount of sample used for degradation by the Schmidt I n this reaction t h e yields of bot,h acetate and carbon dioside were reaction is limited by the flask as two equivalents of gas are nearly quantitative (Table I). formed during t h e reaction (carbon dioxide and nitrogen). With the 50-ml. reaction flasks used here, as much as 0.5 millimole of acet,at,ecan be degraded. Table I. Oxidation of Lactate-Carbon-14 with Oxidation of Methylamine (Equation 3). The vial containing the methylamine was put into a small distillation flask containPermanganate ing a few milliliters of water, and the solution was made alkaSpecific Activitv, line, The contents of the flask were concentrated to a small Counts/hfin./ 7 1x0 volume, and t h e methylamine distillate (about 5 ml.) was trapped . i c t i n t y in Yield, % AT*.^ Carboxyl in an escess of sulfuric arid contained in another combustion Compounda Acetate ~ ~ ~ ~ ~ , ?flask. , One milliliter of 2-V sodium hydroxide and 3 ml. of 5% potassium permanganate were next added, and the flask was 8.5 96.5 97 98 24.6 Lactic acid-1-carbon-14 evacuated and heated for about 30 minutes in an oven set a t Lactic acid-2-carbon-14 99 96 2.4 35.8 2.3 Lactic acid-3-carbon-14 95 96 1.3 18.1 2.4 80" to 90" C. After cooling, alkali was put into the center well, and t h e flask was re-evacuated. The carbon dioxide liberated Obtained from Richard Lemmon, Laboratory, University of by injecting escess sulfuric acid into the main compartment was California. 6 Obtained b y persulfate combustion ( 6 ) of lactate. collected in the manner described above. An alternative procedure for the combustion of methylamine with potassium persulfate can be ueed. I n a previous paper (6) it was reported t h a t t h e carbon dioxide yields from this oxidation Procedure. Two hundred"microm0Ies of lactate were intrawere low. However, amines of this type can be completely oxiduced into the main compartment of the vessel. One milliliter dized, provided a large excess of persulfate is utilized ( 8 ) . When of standardized, carbonate-free alkali was placed in the center well, and immediately thereafter 2 ml. of 5% potassium man200 micromoles of methylamine were being burned, 1 gram of ganate in 2N ~ulfuricacid were added. The flasks were quickly potassium persulfate was found to yield satisfactory results. Simple combustion-diffusion vessels may be used for the complete degradation of carbon-14-labeled lactate and acetate. The results obtained compare favorably with those of other standard methods. This flask is useful for general degradation procedures of carbon-14labeled compounds.
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Table 11. Degradation of Acetate-Carbon-14
also found suitable for the degradation of propionate and butyrate. I t is likely that a variety of degradations can be carried out by means of the simple apparatus described here, with a considerable saving of time. I n this laboratory the complete degradation of serine by the periodate method of Sakami ( I 2 ) was performed in the combustion-diff usion vessel.
Products of Reaction Per Cent Recovery Specific ActivityU Compound Reaction C o t CHaNH2b CO2 CHnNH2c Acetate-1-carbon-14 K&Os oxidation 96 .. 22.7 ... 92 90 46.0 0 dzidereaction Acetate-5-carbon-14 KgSaOs oxidation 96 .. 18.2 ... Bzidereaction 90 91 0 35.9 Acetate-cnrbon-14d K&Os oxidation 90 .. 54.7 ... LITERATURE CITED Azidereaction 95 93 34.7 79.9 an end-windol* ~ ~ i ~ ~ ~ - (1) i f .ironoff, a l ~ S., ~ ~Barker, H. A., and Calvin. If.,J . B i d . Chetn., 169, a Counts/min./mg. B~CO, tube. 459 (1947). b Determined by titration of volatile base. (2) Baker, N.,Feinberg, H., and Hill, It., A N ~ LCHEM., . 26, 1504 C Oxidized t o COI with KiInO4. (1954). d Biologically prepared ( 4 ) , (3) Barker, H.A . , and Kanien, 31. D., Proc. ,\htZ. Acud. Sci.,C. S., _
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Methylamine can also be combusted directly after the Schmidt reaction, without distillation. T h e contents of t h e vial are transferred, with water, to the main compartment of a reaction vessel, and the combustion is conducted as previously described (6). This direct combustion greatly simplifies the procedure. However, if any unreacted acetate is present, i t also will be oxidized. Table I1 shows that the recoveries of both carbon dioxide and methylamine were about 90%. The specific activities of the labeled carbons of either acetate-1-carbon-14 or acetate-2carbon-14 were twice the average values obtained by persulfate combustion. It appears that there is no significant cross-contamination by this method. The advantages Of this procpdure are especially apparent when many samples are degraded simultaneously. The method was
31,219 (1945). (4) Dauben, W.G..Abraham, S.,Hotta, S., Chaikoff, I. L., Bradlow, H. L., and Soloway. -%. H., J . A m . Chem. SOC.,75, 3038 (1953). ( 5 ) D a w L.,XIeinke, AI., and Calvin. AI.. J . BioL Chein., 196, 77 (1952). ( 6 ) Katz, J., Abraham, S.,a n d Baker. S . . .%VAL. CHEM.,26, 1503 (1954). (7) RIarkham, R., Biochem. J . , 36,790(1942). Arch. Biocheilz., 33,IT3 (lg51). Pharesg E. (9) Phares, E. F., personal (10) Roseman. S.,J . Am. Chem. Soc.. 75,3854 (1953). (11)Sakami, w., J. C h e m . , l g 7 9 369 (1950). (12) Wood, H. G., Lifson, x., and Lorher. v., Ibid., 159,475 (1945). RECEIVED for review April 19, 19.54. Accepted October 4, 1954. Supported by a contract from the C. S.Atomic Energy Commission. Work performed during the tenure of a postgraduate fellowship of the American Cancer Society by Joseph Ratz.
Argentimetric Procedure for Borohydride Determination HERBERT C. B R O W N and ALFRED C. BOYD, JR. Department of Chemistry, Pordoe University, Lafayette,
I n order to avoid errors arising from loss of borohydride under acidic conditions, an analytical procedure w-as developed which permits the determination of borohydride under alkaline conditions. The analysis is based ethylene-+ upon the reaction: 8Ag+ BH,8 0 H - diamine
+
+
+
+
8Ag 4 HtBOs5H10. The precipitated silver is removed by filtration and the excess silver ion in the solution is determined by standard volumetric procedures. The procedure allows the determination of borohydride in the presence of iodate and shows that the reaction of iodate with borohydride is very slow. No interference occurs with potassium chlorate, sodium formate, ethyl alcohol, acetone, and cyclohexanone. Benzaldehyde interferes, resulting in high borohydride values. -4n ammoniacal solution of silver nitrate provides a convenient sensitive spot test for borohydride solutions.
I
S THE original investigations of the chemistry of the borohydrides, the determination of active hydrogen utilized hydrolytic dccomposition of the borohydride ion under acidic conditions, followed by measurement of the hydrogen evolved ( 4 , 5 , 9 ) . Volumetric methods based upon the oxidation of borohydride by iodine ( 7 ) ,hypochlorite ( 3 , 8). and iodate (6) have been proposed. The iodate method is a simple procedure which appears to have many advantages for the rapid analysis of borohydride solutions. An exes2 of standard potassium iodate solution is added to the aqueous borohydride sample, stabilized by alkali. A large excess of solid potassium iodide is added, followed by 1 S sulfuric acid.
Id. hfter 2 to 3 minutes in the dark, the liberated iodine is titrated with standard sodium thiosulfate solution. An attempt was made to piniplify the iodate procedure by eliminating the need for two standard solutions. Lyttle et a2. had stated that the borohydride reduction of iodate appcars t o be an instantaneous reaction (6). It therefore appeared that the analysis could be carried out by the Liddition of a large unknown excess of iodate to the l)oroh>.dride solution, conversion of the iodide (presumably formed in the rapid reduction) to free iodine by acidification, followed i,- titration of the free iodine by standard arsenious oxide under controlled pH. The results obtained wcre errntic. In the course of investigating the cause of the difficulties, i t became evident that the reaction of iodate with horoh>.dride is not 30 fast as i t had been postulated to be. The iodate procedure must depend upon the formation of iodine or other int#ern~ediateoxidation productp, upon the acidification of the iodate-iodide solution, followed by the reaction of these products with the borohydride. I n the course of this study an argcntimetric procedure for borohydride determination was devcloped which permits the analysis of borohydride solutions under trongly alkaline conditions and the determination of borohydr d~ in the presence of iodate. Application of the method definitely established the slowness with which iodate and boroh>.dride react under alkaline conditions. The new analytical procedure deppnds upon the reduction of silver ion by borohydride ion under alkaline conditions. I n order 8AgT
+ BHI- + 80H-
+
8.4g
4
+ HzBOa- + 5Hc0
to maintain the silver ion in solution under alkaline conditions,
