ANALYTICAL CHEMISTRY
1706 (8) Frits, J. S., and Fulda, 11.O., ANAL.CHEM.,25, 1837 (1953). (9) Levi, L., and Farmilo, C. G., Ibid., 25, 909 (1953). (10) Levi, L., Oestreicher, P. M., and Farmilo, C. G., United .\‘ations Bull. Xarcotics, 5, 15 (1953). (11) Napoli, J. A., and Schmall, l I . , ANAL.CHEM.,23, 1893 (1951). (12) Perriarowski, M.,Drug Standards, 21, 189 (1953). (13) Pifer, C. W., and Wollish, E. G., AXAL.CHEM.,24, 300 (1952). (14)
Pifer, C. W., Wollish, E. G., and Schmall, M.,J . Am. Pharm.
Assoc., Sei. Ed., 62, 509 (1953). (15) Riddick, J. A., ANAL.CHEM.,26, 77 (1954). (16) Ryan, J. C., Yanowski, L. K., and Pifer, C. W., J . Am. Pharm. Assoc., Sci. Ed., in press,
(17) Schivizhoffen, E. v., and Dam, H., Z . anal. Chem., 140, 81 (1953).
(18) Schnidl, M.. Pifer, C. W., and Wollish, E. G., ANAL.CHEM., 24. 1446 (1952). (19) Schmall, M.~,Wollish, E. G., and Galender, J., J . Ana. Pharnf. Assoc., Sci. Ed., 41, 138 (1952). (20) Siggia, S., Hanna, J. S.,and Kerrenski, I. R., ANAL.CHEM., 22, 1295 (1950). (21) TomiOek, 0.. Stodolovh, A.. and Herman, M.. Chem. Listu. 47, 516 (1953). (22) TomiEek, O., and Vidner, P., Ibid.. 47, 521 (1953). (23) Wagner, C. D., Brown, R. H., and Peters, E. D., J . Am. Chem. SOC.,69, 2609 (1947). (24) Wollish, E. G., Colarusso, R., Pifer, C. W., and Schmall, AI., ANAL.CHEM.,26, 1753 (1954). RECEIVED for review July 29, 1954 .4cceptcd August 25, 1954.
END OF SEVENTH ANNUAL SUMMER SYMPOSIUM
FISHER AWARD LECTURE Wet Carbon Combustion and Some of Its Applications DONALD D. VAN SLYKE Brookhaven National Laboratory, Upton,
N. Y.
.4 partial review of the development of the wet carbon combustion is presented. When either chromic or iodic acid is used alone, many organic substances evol5e part of their carbon as carbon monoxide, and supplementary combustion of the carbon monoxide with oxygen and copper oxide or platinum catalyst has been required for accurate results. An “anhydrous” mixture of iodic and chromic acids in sulfuric and phosphoric acids has been found to effect complete combustion in 1 or 2 minutes, with results comparable in accuracy to those of a dry combustion. The procedure is free from interference by elements such as the alkalies, sulfur, nitrogen, and halides that require modifications of the dry combustion. Work by a nuniber of authors is reviewed in which this combustion mixture has been used, with determination of the evolved carbon dioxide by manometric measurement of the gas, by weighing as either carbon dioxide or barium carbonate, or by titration. Applications to niicrodeterminations of fats and proteins, of various elements precipitated with organic precipitants, and of radioactive carbon are re\iewed.
\\-hich platinum is used to catalyze the dry combustion. This method was being used for amino acids, with which the combustion part of the analysis was easily completed in 15 minutes, but when an attempt was made to burn lipides, extreme caution and 2 hours were required to avoid low results from escape of carbon monoxide. Attempts to devise satisfactory wet combustions, usually with oxidation by chromic acid, go back nearly a hundred years, and 110 attempt is made a t a complete review. Of the modern methods discussed, only enough details are given to indicate the conditions irith which the different methods can he applied with advantage. The original papers must be consulted for necessary details. PROCEDURES WITH SUPPLEiMENTARY COMBUSTIOW BY HEATED CUPRIC OXIDE
The constantly recurring problem has been to avoid loss of carbon evolved as carbon monoxide gas. The first met,hod to be accepted as accurate by the standards of the organic chemist appears to be that published by Messinger in 1890 (1.5). hlessinger oxidized with a mixture of d f u r i c and chromic acids and then led the gases over heated copper oxide to burn carbon monoxide that escaped the chromic acid. The method was really a combination of the dry and wet combustions. It was esact,,but
T
HE dry combustion of carbon has been developed to such precision and convenience that one may well ask what need there is for an alternative wet combustion. There are, however, some conditions under which the dry combustion offers difficulties and other conditions where the wet combustion offers advantages in speed and convenience or accuracy. For some substances, the dry Combustion requires modification. Explosive materials require special handling. If alkali or alkaline earth metals are present, the procedure must be modified or carbon dioxide will be held back as alkali carbonate. Arsenic, antimony, bismuth, and mercury, if present in the sample, become volatilized and ruin the filling of the combustion tube. Boron and thallium have also been, reported t o interfere. For combustion of biological material, it is often inconvenient to get the sample into a boat for the dry combustion. Occasionally substances have been found to defy accurate analysis by dry combustion; the ease with which complete combustion to carbon dioxide is attained can vary greatly. The author recalls an encounter with this variability when doing combustions in P. -4.Levene’s laboratory with the method of Dennstedt, in
VACUUM
IA
IB
VACUUM
IC
Figure 1. Apparatus of Nicloux, l A , and Ita Modifications by Boivin, IB, and Kirk and Williams, IC
V O L U M E 2 6 , NO. 11, N O V E M B E R 1 9 5 4
1707
it was complicated, and the combustion took 2 hours besides the time for weighings. Kuster and Stallberg (12) in 1893, encountering a triniethyl aromatic compound that gave results about 9% too low by dry combustion, applied Messinger’s combined dry and wet combustion and simplified i t in detail so that the combustion could be hnished in shorter time. Kiister and Stallberg’s procedure is given by hleyer as still the standard wet combustion method for precise results (16 ) . Nicloux’ Method. For carbon determination in biological material. the Messinger-Kiister-Stallberg method is too complicated to be convenient when numerous analyses are required. The wet rombustion has consequently been applied to biological problems by a number of investigators in simplified forms, with omission of the cumic oxide-charged - combustion tube. With m r h procedures, using chromic acid in most cases, it has been possible to obtain quantitative results with many substanres and approximate results with others. Of such procedures, that of Nicloux (181, e1nPlOYing the simplest of apparatus (Figure 1, 1,4), proved especially Practical. The method was designed especially for determination of 1 to 3 nig. of citrbon in 0.3-nil. samples of urine.
gases and facilitate complete combustion. The determination of the carbon dioxide by precipitating as barium carbonate and titrating was as by Nicloux. An analysis required about an hour. Boivin’s development of the Xicloux method to a general one for organic analysis involved a return to Messinger’a principle of using a supplementarv combustion to burn carbon monoxide evolved from the wet combustion. It required as much time as the Nessinger method, but used simpler apparatus. Kirk and Killiams (11) have made Boivin’s method more eonvenient by using a removable short tube, C in Figure 1, I C , to hold the 2N potassium hydroxide used for absorbing the carbon dioside. Instead of washing the barium carbonate by centrifugation, Kirk and Williams wash it on an asbestos filter with saturated barium carbonate solution. PROCEDURES W ITHOUT SUPPLEJfE\TkRY CO\ZBUSTIO\
xicloux used for oxidation a mixture of 6 ml, of sulfuric acid, 0.05 gram of silver chromate, 0.250 gram of potassium dichromate, and 0.6 gram of a11hYdrous sodium sulfate. The solid reagents and 0 3 nil. of aqueous sample are placed in tube D (Figure ), ‘and o,5 ml. of 2Lypotassium hydroxide, to absorb carbon dioxide, is placed in C. The entire apparatus is evacuated through A to 10 or 20 mm. of mercury pressure, and D is immersed in boiling water for a few minutes, evaporat’ing the water of the sample, which is condensed in the bulb B. Five milliliters of sulfuric acid are then admitted from E and bustion i p obt,ained b), heating jyith a microburner until the chromic acid mixture turns green. The oxygen gas evolved by the reaction. 2H~Cr?07, 6HzS04 = 2Cr?(S04)3 -k 302 -k 8H20, drives the carbon dioxide UP into bulb B , where i t is absorbed by revolving the alkali solution. The alkali solution is then Lvashed into a centrifuge tube, where the carbonate i p precipitated as barium carbonate by addition of barium chloride. The barium carbonate is washed by centrifugation, redissolved in 10 ml. of 0.05N hydrochloric acid, and determined by titrLtt,ingthe excess with 0 . 0 5 ~ sodium hydroxide.
+
This method gave consistent results with urine and with similar 0.3-ml. samples of aqueous solutions of a number of compounds. The amount of carbon escaping as monoxide was only of the order of 1 or 2%. Nicloux’ Method with Supplementary Combustion by Oxygen and Heated Platinum. However, Boivin (3) found that the same procedure was not accurate for general analysis of dry samples of organic compounds, because greater amounts of carbon n.ere evolved as carbon monoxide. The quicker burning substances evolved the most monoxide, sugars, for esample, evolving 10 to 1 5 9 of their carbon as monoside. To complete the combustion of the monoxide, Boivin modified Xicloux’ method by introducing a loop of platinum wire into bulb B (Figure 1, 1B). The reagent, for
of silver chromate, and a few decigrams of a n h y d r o u s sodium sulfate. The volume of bulb B was made 50 to 60 ml., sufficient to hold all the oxygen evolved from the excess chromate. To obtain good results it was found necessaryin toa heat the mixture in at water bath for 20 to 30 minutes before finishing the combustion with a gas flame. At the end of the combustion enough 30 volume yo sulfuric acid was admitted from E to fill tube D nearly to the top, driving the gases up into bulb B. Combustion of carbon monoxide to dioxide R-as then completed by heating the wire to a glow 50 or 60 times, the alternate heating and cooling serving to stir the
c.
The problem remained t o devise a wet combustion mixture that lvould in itself burn all the carbon to carbon dioxide quickly. iyithout resorting to a supplementary combustion to burn carbo11 ~h~ need for quick and accurate combustion in studies of lipides and amino acids at’ the Rockefeller Institute led to attempts to solve this problem. I t x-as finally found ( $ 2 ) that when iodic and chromic acids together were used in an approximat,ely anhydrous medium made by mixiRg fuming sulfuric3 with phosphoric acid, combustion was complete in 1 or 2 minutes with a wide variety of compounds, including relatively resiptant substances such as fatty acids and cholesterol. This combustion misture was attained after many discouraging trialp, which a t one time led to reverting to an accessory tube with heated copper oxide. The above combustion mixture, called here the “anhydrous” mixture, was a mixture of 67 ml. of fuming sulfuric acid (20% sulfur trioxide), 33 ml. of phosphoric acid of specific gravity 1,i2, grams of and grams of potassium iodate. -4t room temperature the mixture was supersaturated with respect to iodic acid, but a solution prepared with heat would hold its iodic acid for a .rvorking day. F~~most substances it sufficed to use a stock solution of chromium trioxide in the acid mixture and add pulverized potassium iodate with t,he organic sample in the combustion tube; the iodate dissolved as soon as the chromic acid mixture was heated. Carbohydrates, however, required the presence of iodic acid from the start of the reaction in order to avoid the escape of 1 or 2% of carbon as carbon monoxide. Eventually (26), the oxidizing mixture was modified to the two fornis given in Table I. For a combustion, the sample and “solid reagent” are placed in the combustion vessel and the latter is connected with apparatus to absorb the carbon dioxide. The “liquid reagent” misture of phosphoric and sulfuric acids is then added and the total mixture is heated for about 1.5 minutes after apparent boiling has begun. The “apparent boiling” is in fact evolution of oxygen from the chromic acid. The misture
Table I.
Reagents for Wet Carbon Combustion (26)“
A~~~~~ of Carbon
Reagent Anhydrous general cambustion mixture IIixture for carbohydrates
in Sample, Ilg. 0.2-0.7
1.0-3.5
4-15 0.2-0. i 1.0-3.5 4-1.5
Liquid Reagents.-ALl. Mixture .4nhydrous for carbomivturea hydratesc 2 2 5
.. ..
..
2 2 r,
..
..
Volume a t Whicli Reagentb, Grams cos pressure KlOv KlOr Measured in K a C r ~ 0 7 KaCrlOi Manometric ( 2 t o 1) (10 t o 1) Analy.sis. M1. 0.15 2.0
..
0.30 1 .oo
10.0 4F.0
0 13 0.30
2.0 10.0
1
4ij. 0
.oo
a One of the pulverized solid reagents is placed in romhustiori tube with dry sample. connection is made with vessel t o ahsorb os freeze evolved carbon dioside; t h r n t h e appropriate &id reagent is Rdded and heat applied. b T w o volumes of fuming sulfuric acid (20% sulfur trioxide), 1 volume of phosphoric acid (sp. gr. 1.72). and 1 gram of potassium chlorate per 100 ml. C One volume of sulfuric acid (sp. gr, 1.84), 1 volume of phosphoric arid (rp. pr. 1 . 7 2 ) . and 1.5 grams of potassium chlorate per 100 nil.
1708
ANALYTICAL CHEMISTRY TO SUCTION
Table 11.
Results of 3Ianometric Wcroanalyses (22)
Substanco Stearic arid
Minutes Boiled 1
Sample hIg 4 131
1
4.4fil 4.132
C holes t crol
Silver acetate Pentacetyl glucose
2 2 3
3.975 4.218 3 . ,530 3,233 3 . 174 3.683 3.370 3.709
1 2 3
49.25 49.53 49.33
a
19.050 1R. 703 18.227 4 983 4.834 4,885
75.80 i5.86
3 3 1 2 3
1
1
(‘arbon, 7 ‘* Found Theoretical 75.98 7 6 . no iR.13 75.87 83.98 83.87 84.02 83.91 83.93 83.99 14.35 1.1.39 14.40
1 2
r7-
83 67
lL3i
49.23
B
McCREADY AND HASSID
Figure 3. Apparatus of McCready and Hassid for Absorbing Carbon Dioxide in -4scarite in Gravimetric &-et Combustion
Table 111. Results of Combustion with Anhydrous Mixture Followed by Absorption and Weighing of Carbon Dioxide in Soda Lime Tube (14) Carbon. % Sample, COz. Substance Glncose 2,3,4,6-Tttranicth3-1@-methylglucoside Starch acetate Palniitir acid
f
Phcnq-lhq-drazine hydrorhloride Benzoic w i d
1
Figure 2. -4rrangement of Van Slyke and Folch for Combustion with .4bsorption of Carbon Dioxide by Alkali Solution in the Manometric Apparatus of Van Slylte and Neil1 -4bsorbed carbon dioxide in chamber C is subsequently freed b y acid and measured b y the pressure exerted at 2 - , IO-, or 46-ml. volume for amounts of carbon in the ranges 0.2 to 0.7, 1 to 3.5,and 5 t o 15 mg., respectively
does not reach the boiling point of the acids themselves. For the reagent for carbohydrates, in which the solid reagent is 90% iodate, the 1 to I mixture of ordinary concentrated sulfuric and phosphoric acids is a medium preferable to the anhydrous acid mixture, because the boiling temperature of the former does not decompose iodic acid to free iodine, and the anhydrous condition is not required. I n the “anhydrous reagent” for general combustions enough excess chromate is present to prevent evolution of iodine unless heating is unnecessarily prolonged. As long as chromic acid is present, any free iodine formed is oxidized back to iodic acid. It is undesirable and unnecessary to continue heating the anhydrous reagent until the chromic acid has lost oxygen and evolution of iodine has begun. Evolved iodine does not cause error in the analysis, but is inconvenient because i t can eventually clog connecting capillaries.
11g.
23.82 11.22 15.30 10.65 24.00 6.48 7.87 14.40 27.34 12.90 13.30 11.18
lfg. 33.40 16.50 29.47 20.50
44.20 11.85 21.61 49.58 49.76 23.48 33.40 28.20
Found 39.91 40.11 82.53 a2.49 50.22 49.87 74.88 74.95 49.63 49.65 68.51 65.78
Calculated 40.00 52.78 50.00
7.1.94 49.52 68.85
The first procedure ( 2 2 ) employed 17-ith the new reagents was to absorb the carbon dioxide Tyith a solution of sodium hydroxide and hydrazine in the manometric apparatus of Van Slyke and Neill. The absorbed carbon dioxide was then set free by addition of acid and was measured manometrically. This is still routine in this laboratory. A similar procedure with this apparatus, but with chromic acid oxidizing mixtures of more limited application, was first employed by Backlin ( 1 ) in 1930 in Sweden, and by the writer, Page, and Kirk (24). It has been modified with regard to construction of apparatus as well as reagents (22, 26).
Table IV. Results of Combustion with ..inhydrous 3Iixture followed by Absorption of Carbon Dioxide in Sodium Hydroxide, Precipitation as Barium Carbonate, and Measurement of the Barium Carbonate by Titration (5) Carbon, M g Sodinm ovalatc
Present 2 92 2 15 1 87 1 80 1 na 1 88
Potassium hydrogen phthalate
4.51
29 5.33 ti
Li,f31
p,B’-Dichloroethyl sulfide
2 73
hdipic acid
5.84 5.38 5.20 5.47
Found 2 95
2 16 1 84 1 88 1 68 1 02 4.54 6.26 5.33 5.61 2.7G 5.85 5.43 5.20 5,51
V O L U M E 2 6 , NO. 1 1 , N O V E M B E R 1 9 5 4 The dry sample and solid reagent (Table I ) are placed in combustion tube T (Figure Z), which is then connected with the manometric chamber, C, as shown in Figure 2. Chamber C is partially evacuated and 2 ml. of 0.5S sodium hydroxide, cont,aining hydrazine to reduce halides, is admitted from E . Two milliliters of liquid reagent (Table I ) are run into T, and the niixt,ure is heated until it has boiled for 1 to 2 minutes. The carbon dioxide evolved is absorbed in the alkali of C by raising and lowering the mercury. The combust,ion tube is then disconnected, unabsorbed gases (oxygen and nitrogen) are ejected from C and acid is added t,o liberate the absorbed carbon dioxide. The liberation is complet,ed by shaking the partially evacuated chamber for 2 minutes with the mercury a t the 50-ml. mark a t t,he bottom. The carbon dioxide gas is then brought to a definite volume, 2, 10, or 36 ml., depending on t,he size of the carbon sample (see Table I ) and the pressure exert'ed by the gas is measured on the nianomet,er. Combustion and manometric measurement can be completed in 15 minutes. With the same apparatus one can do a submicro analysis with 0.2 to 0 . i mg. of carbon, a microanalysis with 1 to 3 nig., the amount used in the usual dry microcombustion, or :isemimicro analysis with 5 to 15 mg. of carbon, putting the gas a t different volume marks in the manometric chamber for the pressure measurements. \Vhen the sample is in solution, either in n-ater or in a volatile organic solvent, an aliquot is evaporated t o dryness in the combustion tube, and the analysis is performed on the residue (82). As indicated by Table 11, the error is usually less than 0.3y0 of the amount of carbon determined when the analysis is made on samples containing 2 t o 3 nig. of carbon, the carbon dioxide
AI
1709 being measured by the pressure that it exerts a t 10-ml. volume. When submicro analysis are done with samples of 0.3 to 0.7 mg. of carbon and measurement of the carbon dioxide pressure a t 2-ml. volume, the usual limit of error is about +0.5%. The completeness with which the anhydrous combustion mixture ( 2 2 )burns organic carbon to carbon dioxide has been checked by other analysts using other methods to measure the carbon dioxide. hIcCready and Hassid ( 1 4 )absorbed the carbon dioxide and weighed it, as in dry combustions. Their apparatus is shoxn in Figure 3. The gases from the combustion vessel, B, are passed through condenser C, then through zinc tower, F , to remove acid or halogen, then dried in G n i t h magnesium perchlorated, and finally passed into tube H , where the carbon dioxide is absorbed by Aecarite (asbestos impregnated Kith sodium hydroxide). I is a guard tube viith dry calcium chloride. The results shown in Table I11 are of such accuracy that these investigators state that the method is as accurate as the classical dry combustion. I n Figure 4 is shown an arrangement, extreme in its simplicity, used by Lindenbaum, Schubert', and Armstrong ( 1 3 ) for gravimetric measurement of carbon dioxide formed in combustion tube E yielded by the "anhydrous" combustion mixture. The carbon dioxide in this case is absorbed in barium hydroxide solution in H . The tn-o chambers are evacuated before the acid of combustion mixture is added from B. The oxygen evolved from the mixture by continuing the heating after the combustion proper is over serves t o drive the last portions of carbon dioxide over into the barium hydroxide solution. The bwium carbonate precipitate is weighed. TWh semimicro samples of 10 to 15 mg. of carbon, results similar in accuracy t o those of JIcCready and Hassid were obtained. Titration of the carbon dioxide liberated bj- the anhydrous wet combustion has also yielded theoretical results accurate within the limits of the titration procedure. I n a method developed by Farrington, Siemann, and Sn-ift ( 5 ) , the carbon dioxide is absorbed in sodium hydroxide from x-hich it is then precipitated as barium carbonate. Adherent alkali on the centrifuged barium carbonate is neutralized by addition of 0.055 hydrochloric acid until the red color of phenolphthalein disappears. Then the barium carbonate is determined by dissolving it in excess 0.0LV hydrochloric acid and titrating back the excess hydrochloric acid with 0.05A' alkali. The preliminary neutralization of adherent sodium hydroxide obviates the necessity of washing the barium carbonate. Some of the results obtained are shown in Table IIr. APPLICATIONS TO BIOLOGICAL MATERIAL A N D DETERMINATIOSS OF ELEMENTS OTHER THAN CARBO3
D
F
E H
Figure 4. Apparatus of Lindenbaum, Schubert, and Armstrong for Wet Combustion with Collection of Carbon Dioxide for Gravimetric Determination as Barium Carbonate II is charged with barium hydroxide solution. Dry sample and potassium iodate are placed in E. E and H are evacuated. Chromic acids dissolve i n sulfuric and phosphoric acids is admitted through C .
I n addition t,o elementary analysis of pure organic substances, the wet combustion method affords the most precise micromethod for the total fats of blood serum. The procedure now used a t Brookhaven ( 2 5 ) is as follows: Prot.eins and fats are precipitated together by zinc hydroxide after dilut,ing the serum with 25 volumes of ivater in a centrifuge tube. The precipitate is washed twice with water in the centrifuge tube to complete the removal of water-soluble extractives. The Tvashed residue is then mixed with enough 2 to 1 chloroformmethanol lipide solvent, introduced by Folch ( 6 ) , t o make the total volume 25 times that of the serum sample, and the protein precipitate is centrifuged down. dliquots of the supernatant lipide solution are measured into conibust'ion tubes, dried, and the carbon is determined in the residue. The 0.5 nig. of fat from 0.1 ml. of normal serum suffices for an analysis precise wit,hin 1 part in 200. The determination of lipide carbon is analogous to that of protein nitrogen. hnother microdetermination is that of serum proteins, introduced by Hoagland (9), who preferred it to the micro-Kjeldahl, Proteins in a sample of 0.02 ml. of serum are precipitated in the centrifuge-combination tube (Figure 5) with alcohol-ether, and are washed with the same solvent to remove lipides. The protein precipitate is then dried and the carbon is determined. The nianometric wet combustion has been used in niicrodeter-
ANALYTICAL CHEMISTRY
1710 Table
\-.
inder which, after weighing, is placed under the window of a Geiger counter. Smount The manometric wet combustion of of Element total carbon has been combined, a t the Required for a n Analysis, Brookhaven National Laboratory ( d 7 ) , arg. Authors with counting either as barium carbonate 0 01 Kirk (10) Hoagland ( 8 ) or as carbon dioxide gas, to yield simul0.03 Hoagland (7) 0.03 Hoagland (8) taneous determination of total carbon 0.02 Van S b k e and K r e w (2% and its carbon-14 specific activity, -4fkr 0.02 Thompson (SI) the carbon dioxide is measured in the -. . . manometric apparatus, it is transferred either to a barium hydroxide solution or directly to a gas counter. For the barium carbonate counting, a test tube containing 2.5 ml. of barium hJdroxide of approxiniately 0.25iV concentration is attached to the manometric chamber, as shown in Figure 6. The air in the tube 2nd in the connections is evacuated with a water aspirator, and the carbon dioxide from the chamber is passed into the tube and absorbed by the barium hydroxide. The carbonate is then filtered on a porous glass disk and used for counting. For transfer of carbon dioxide gas directly from the manometric chamber to a gas counter, the arrangement is shown i n Figure 7.
llicromineral 411al) ses by Combustion of Organic Precipitates
Illeluent Pliosphoriis
T o . of Carbon Atoms for One Atom of Precipitate Element 73 Strychnine phosphomolybdate
3Jagnesiiim Sulfur Calcium Sodium
Hydroxyquinolate Benzidine sulfate Picrolonate r r a n y l zinc acetate
19 12
20 20 ~
mm .
!
c+
I
llmm (outer diameter)
Figure 5. Combustion Tube for Washing and Combustion of Small Precipitates (7)
The gas counter, E, from Bernstein and Ballentine (a),is a cylinder of approximately 100-ml. volume with a wire anode in the center: a silver coating about the interior wall serves as cathode. The glass coil, D, in the connecting tube is immersed in a cooling liquid of dry ice and alcohol to intercept moisture. The counter tube, E, is partially immersed in liquid nitrogen to condense carbon dioxide. After the carbon dioxide obtained in a combustion has been measured manometrically, the manometric chamber and the counter are attached as shown in Figure 7 , exccpt that the cock of the chamber is closed as in Figure 8, instead
niination of several elements which form precipitates with organic reagents. The precipitations are made in centrifuge-combustion tuhcs (Figure 5) in which the precipitates are washed, dried, and burned. Table V indicates a number of analyses that have been made by this procedure. COMBINED DETERMINATION OF CARBOX AhD ITS RADIOACTIVITY
During the past few years the “anhydrous” wet combustion mixture (22, 2 6 ) has attained fairly general use in determining the radioactivity of organic mixtures containing carbon-14. The carbon dioxide is counted either as carbon-14 labeled barium carbonate or carbon dioxide gas. When radioactivity is high enough to give accurate counts of carbon-14 as barium carbonate, the carbonate procedure has an advantage in that the disk arrangements used for collecting and counting the carbonate are cheaper than the counting tubes and accessories used for the gas. -41~0,when only specific activity is desired, it is not necessary with barium carbonate counting to determine accurately the amount of carbon in the sample. With a barium carbonate layer of “infinite thickness”-viz., 20 mg. or more per square centimeter-the count is independent of the thickness. The carbonate, hon-ever, is less fitted to measurement of weak activities, because only a small percentage of the disintegrations is counted; a large part of the beta rays is absorbed before they escape the barium carbonate, and another part is lost by the geometry of the counter. Ten per cent or less may get counted. More efficient measuring of weak activities is provided by transferring the carbon dioxide in the gaseous form to either a proportional counter or an ionization chamber. In the gas counters there is no loss from self-absorption and but little from geometry. The method of Lindenbaum, Schubert, and iirmstrong (IS) (Figure 4)is one of many in which the carbon dioxide yielded by combustion with the anhydrous reagent is converted into barium carbonate that can be used for counting carbon-14 activity. The carbonate is collected with wction in a shallow filtering cyl-
c
G
Rubber Bands
i
I
Figure 6. Manometric Chamber Arranged for Transfer of Carbon Dioxide to Barium Hydroxide Solution Preliminary to Carbon-14 Labeled Barium Carbonate Counting (27)
V O L U M E 26, NO. 11, N O V E M B E R 1 9 5 4
ber is ouened as in Fieure f. 'The caibon dioxide passes rapidly
integrations of carbon-14. With this procedure it iipossible to measure specific carbon-14 activities as little as 1%of those that can he measured in the same counting time with the same BCCU-
:
iK
1711 For extremely weak specific activities, with material of which large amounts are available, Buchman's arrangement has over that of Van Slyke, Steele, and Plsain an Sdvantage in that the size of sample employed in Buchanan's apparatus can he 15 times greater. The background is about five times greater (450 vs. 80 counta per minute). It can be calculated [Equation 9 (27)]that to obtain a count with &30/, standard deviation, in a total time of 100 minutes for sample and background counts, the equipment of Van Slyke, Steele, and Plaain with a 15-mg. cmhon sample would require %. disintegration count of 4.8 per minute per milligram of carbon; while with Buchanan's procedure and a 225-mg. carbon sample an activity of 0.7 count per minute per milligram of carhon would suffice, or one sevrnt,h as great 8, specific activity. For microanalysis, with 0.1 to 15 mg. of carbon, the Brookhaven arrangement (27) is more accurate and convenient. It also bus the advantage that it can be set up in a feu. minutes on ordinary lahoboratory bench in connection with the standard V : L ~ Slyke-Neil1 manometric apparatus, which is used for various other analyses when not employed for carbon-14. I n this l a b oratory, there are four or five difcrent manometric apparat,uscs that are a t times used for casbon-14 and a t other times for vwious other manometric analyses. At the Oak Ridge Laboratory, the completeness of the comhustion of organic substances by the anhydrous wet combustion mixture is taken for granted in %. technique that is used for measuring t,he carbon-14sotivity of pure substsnces. The substance isburned in tlre anhydrous combustion mixture and the carbon dioxide is transferred without measurement directly to a previously evacuated ionization chamber nhere the activity is measured. The total carbon is assumed to he that theoretically yielded by was introduced in 1948 t.he substance burned. This procedure . . .... . .
1712
ANALYTICAL CHEMISTRY Kuster, F. W., and Stallberg, A., A m . , 278, 215 (1893). Lindenbaum, d.,Schubert, J.,and Armstrong, W. D.,. ANAL. CHEhl., 20, 1120 (1948). XIcCready, R. >I., and Hassid, W.Z., IXD. ENG.CHEY.,;INAL. ED., 14, 526 (1942). Nessinger, J., Ber., 23, 2756 (1890). IIeyer, Hans, “Organic Analysis,” Berlin, Julius Springer, 1938; .bin Arbor, Mich., Edwards Bros., 1943. Neville, 0. K., J . Am. Chem. Soc., 70, 3499 (1948). Sicloux, 31.RI., Bull. soc. chim. biol., 9, 639 (1927). CHEM.,25, 174 (1953). Raaen, V. F., and Ropp, G. A , -4s.~~. Sinex, F. AI., Plasin, J., Clareus, D., Bernstein, W., Van Slyke, D. D., and Chase, R., in press. Thompson, W.B., K. Y. State Dept. Health, Ann. Rept. Diu. Labs. and Research, 1945, 21. Van Slyke, D. D., and Folch, J., J . Biol. Chem., 136, 509 (1940). Van Slyke, D. D., and Kreysa, F., Ibid., 142, 765 (194.5). Van Slyke, D. D., Page, I. H., and Kirk, E., Ihid., 102, 635 (1933). Yan Slyke, D. D., and Plaain, J. P., unpublished work. 1-an Slyke, D. D., Plasin, J. P., and Weisiger, J. R., J . Biol. Chem., 191, 299 (1951). Van Slyke, D. D., Steele, R., and Plasin, J. P., Ibid., 192, 769 (1951).
counter. For such, since the combustion itself requires only a few minutes, the procedure makes combustion of many samples per day possible. The standard error of 0.2 to 0.3% in specific carbon-14 activities determined by Raaen and Ropp indicates the reproducibility of both the counting procedure and the combustion with the “anhydrous” mixture. ACKNOWLEDGMENT
The author acknowledges the generous support of Eli Lilly and Co. LITERATURE CITED
(1) Backlin, E., Biochem. Z., 217, 483 (1930). (2) Bernstein, W., and Ballentine, R., Rev. Sci. Instr., 20, 347 (1949). Bull. soc. chim. biol., 11, 1270 (1929). (3) Boivin, 9., (4) Buchanan, D. L., J . Am. Chem. Soc., 74, 2389 (1952). (5) Farrington, P. S., Kiemann, C., and Swift, E. H., ; ~ N A L .CHEM., 21, 1423 (1949). (6) Folch, J., dscoli, I., Lees, AI., Neath, J. A , and LeBaron, F. S . , J . Biol. Chem., 191, 833 (1951). (7) Hoagland. C. L., Ihid.. 136, 533 (1940). (8) Ibid., p. 543. (9) Hoagland, C. L., and Fischer, D. J., Proc. Soc. Ezptl. Biol. M e d . , 40, 581 (1939). (10) Kirk, E., J . Biol. Chem., 106, 191 (1934). . (11) Kirk, P. L., and Williams, P. A., IND.ENG.CHEY.,d s a ~ ED., 4, 403 (1932).
RECEIVED for review August 28, 1953. Accepted July 19, 1954. Presented before the Division of Analytical Chemistry at the 123rd Meeting of the A a a ~ n r r . 4CHE?.IICAL ~ SocmTr, Los dngeles, Calif., I\.lrtrch 1953. Research done under the auspices of the Atomic Energy Commission.
Application of Statistical Analysis to Analytical Data P. D. LARK School o f Applied Chemistry, The N e w South W a l e s University o f Technology, Broadway, Sydney,
The principle of least squares provides a means of separating and estimating the systematic and random errors in determinations by an analytical method if it is investigated over a sufficient range of concentrations. The method of obtaining the estimates of error and interpreting them depends on the adoption of some suitable hypothesis about the relationship of the tw-o types of error to the amount of substance being assayed. Unless this is done, tests of significance or statements involving probability ma>-not be made. In the extended treatment of an example the necessary steps in a statistical examination-choice of hypothesis, computation of regression equations and associated errors, and testing for rejection of suspected values-are illustrated. The results of such an examination are applied to the prediction of the true amount of substance in a sample from that found by chemical analysis.
I
IT T H E investigation of a method of quantitative analysis i t
is desirable to carry out tests over a range of concentrations or weights of the substance analyzed. For any given amount of substance taken the amount found n-ill vary, and the difference or error may be divided into a random part, the cumulative result of steps in procedure and measurement, and a systematic part, sometimes a constant error or blank, which is the fault of the method. Statistical analysis of regression, which may be carried out if the range of the investigation is reasonable, provides a means of separating these two sources of error and enables the reliability o€ the method to be assessed as precisely and accurately as the data permit. -4n example of the application of regression analysis t o data of this type is presented by Youden (‘IS). I n this example, the dependent variate, Y, is the amount of substance found, the independent variable, X , is the exactly known amount taken, and an equation, f‘ = a b X , is fitted by the method of least squares. Here is stated ( I S ) and in a previous paper ( I ? ) and note ( 1 1 ) that
+
N.S.W., Australia
the intercept, a,in this equation is “an estimate of any constant error,” and that there is no evidence of constant error or blank in the analytical process if a does not differ significantly from zero. If,in addition, the slope, b, does not differ significantly from unity, i t seems to be implied that there is no systematic error in the method but only the random error of analytical determinationbX non- indicates the ideal relationship, Y = X. Le., ? = a However, while this may sometimes be true, a slightly more elaborate treatment reveals a different state of affairs in the example discussed. The position becomes clearer if the regression oi total error of analj tical determination, Z = Y - X , on amount taken is considered. The alternative and more instiuctive equation takes b’X, in which b‘ = b-1, and which has a the form: Z = a standard error of estimate, s(e), equal to that of the equation of Y on X . To interpret the constants so obtained, i t must be assumed that this equation estimates a true relationship of the same form. T o be more precise, the hypothesis is made that the systematic error, Z’, varies and is related to X by a linear equation, Z‘ = 01 p‘X, and that the random error, E , is normally distributed with constant standard deviation, u. TTe may write
+
+
+
z = Z’ + E
=
cy
+ j3‘X +
E
for total elror equals Eystematic error plus random error. *$ecepting this, Z‘, 01, p’, and u are best estimated by ,$?,a, b’, and s(e). S n alternative hypothesis might be adopted, that there is no p’ and that the systematic error is a constant, Z‘ = a’’. If this is so, no trend should be shonn by the Z values, and the slope b’ should be close to zero and b to unity. Thus, if the reasonableness of either hypothesis is to be judged from the data themselves, i t is the value of b‘, or of b, that is the criterion of constant or other systematic error, while tests on a have a confirmatory value only. Furthermore, a and b are not independent and tests of significance must be made m-ith due care. These two do not exhaust the number of feasible hypotheses which might be made, although they are the most practical.
