THE DETERMINATION OF CARBON AND CARBON DIOXIDE.

BY THEODORE E. FRIEDEMANN AND ARTHUR I. KENDALL.

(From the Department of Research Bacteriology, Northwestern University Medical School, Chicago.)

(Received for publication, August 25, 1928.)

One of the desirable additions to the armamentarium of chem- istry is a rapid, simple, and precise method for the determination of carbon, At the present time there seem to be but two out- standing methods for this purpose, the dry combustion method of Liebig and the so called Messinger (l-3) method, the latter per- mitting of combustion in the wet way.

The dry combustion method does not lend itself well to organic substances in solution, and it is frequently difficult or impossible to remove liquids by feasible procedures, leaving all of the carbon behind in the residue in suitable condition for analysis. This applies particularly to some of the materials of biochemical im- portance, as for example, urine, blood, tissues, and bacterial culture media. Desiccation of the materials enumerated in- variably is accompanied by loss of CO2 and some of the other more volatile substances, such as alcohol and the lower fatty acids. For such complex mixtures the wet combustion method would seem to be preferable, since the oxidation may be carried out without the preliminary removal of water. It is found, however, that water which is present in the material analyzed interferes in the oxida- tion of many substances by the chromic-sulfuric acid mixture. This is particularly true of such substances as acetic acid and the fats. To obviate this difficulty, the oxidation with chromic- sulfuric acid in the presence of silver (4) or mercury (5) salts to act as catalysts, has been suggested.

It is significant that the majority of favorable results recorded in the literature (3, 6-S) has been obtained when the acid con- centration is relatively high, and the volume of water small, or

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46 Carbon and Carbon Dioxide

even absent. On the other hand, it is equally significant that at least a majority of unsuccessful attempts to use the method has been those in which the amount of acid is relatively small, and the volume of water relatively large (9, 10). These discrepancies, due presumably to the water present, have suggested the work here presented. A study has been made of the effect of water and of acid upon the precision of the wet combustion method. The results obtained show quite convincingly that both acid concentration and volume of water affect the precision of the procedure. When proper compensation is made for t’he water present the carbon content of even such difficultly oxidizable substances as acetic acid and fats may be determined with satisfactory results.

The apparatus described consists of a reaction flask, a reflux condenser, and an absorbing system for COS. It is essentially the apparatus described by Messinger (3), in 1890, but simplified, and in some of its details is like that of Ames and Gaither (10). The oxidizing reagents consist of chromic, sulfuric, and phos- phoric acids, used in the proportions recommended by Schollen- berger (11). Carbon dioxide resulting from the oxidation, is absorbed by 0.5 N NaOH in a tower of special construction and determined by titration with 0.5 N HCI after addition of an excess of BaClz (12).

Description of Method.

Apparatus.

The construction of the apparatus is shown by Fig. 1. A single unit is shown; it is convenient and economical to have several units mounted on a common frame.

A is a 300 cc. Kjeldahl flask fitted with a rubber stopper through which pass Tube B, and C, a Hopkins condenser. Tube B fulfils two purposes: the separatory funnel at the top permits of the addition of the oxidizing mixture; the side arm is connected by rubber tubing with the carbon dioxide-free air main. The Hopkins condenser connects by rubber and glass tubing with the flask, D, of 500 cc. capacity. Emerging from flask D is the absorption tower, E, containing glass beads. The latter are held in place by a perforated porcelain plate, and are solid and of two sizes, respectively, 2 and 3 mm. in diameter. The absorption tower is connected in turn with the tube, F, partly filled with water. This is a safety tube, aIso indicating the

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T. E. Friedemann and A. I. Kendall 47

rate of flow of the carbon dioxide-free air through the apparatus. This is regulated by clamp G, on the rubber tubing which joins

FIQ. 1. Apparatus for determination of total carbon and carbon dioxide.

the tube, F, to the vacuum pump. The vacuum pump draws a current of carbon dioxide-free air from the main through the side tube in B to flask A. From here it passes, together with

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48 Carbon and Carbon Dioxide

carbon dioxide liberated during the digestion, to the Hopkins condenser, where water vapor is condensed, and falls back into flask A. The carbon dioxide and air pass to flask D, and up- wards through the absorption tower, which contains a definite amount of standardized caustic soda. During its passage through the tower, the carbon dioxide is absorbed by the alkali, and the residual air passes away through tube F to the vacuum pump.

The entire apparatus is suspended by means of two burette clamps. These permit of sidewise movement, a point of impor- tance in attaching flasks A and D. The frame for the apparatus is made of t inch iron pipe, joined by elbows and T’s. It is at- tached to the desk by flanges.

Carbon dioxide-free air is supplied through two 5 foot towers made of 2 inch iron pipe. The ends are capped, and tapped to fit 4 inch nipples. These make convenient attachments for pres- sure tubing connecting with the air mains of the apparatus. These towers are filled with moistened soda-lime, about equal portions of 4 and 8 mesh.

Micro burners, connected to the gas mains through g inch valves, provide satisfactory and adjustable sources of heat.

Reagents.

1. Chromic Acid.-340 gm. of chromic acid (CrOa) are dis- solved in 400 cc. of hot COz-free distilled water and made up to a volume of 1 liter with 85 per cent phosphoric acid.

2. Sulfuric-Phosphoric Acid.-Equal volumes of concentrated C.P. sulfuric acid and 85 per cent phosphoric acid are mixed, cooled, and kept in a glass-stoppered bottle.

3. Standard NaOH Solution.-An approximately 0.5 N solution. 4. Standard HCI Solution.-An exactly 0.5 N solution. 6. Phenolphthalein.-A 1 per cent alcoholic solution. 6. Molar Barium Chloride Solution. 7. COz-Free Distilled Water.-Distilled water is boiled 10 to

15 minutes and allowed to cool.

Procedure.

A sample containing from 25 to 100 mg. of carbon is weighed or measured in the 300 cc. Kjeldahl flask and the flask is connected to the apparatus. Next, 75 cc. of standard alkali are measured into a 500 cc. Erlenmeyer flask and connected to the tower. The

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T. E. Friedemann and A. I. Kendall 49

aeration is started and regulated by clamp G so that air passes through tube F at the rate of 50 to 150 bubbles per minute. 10 cc. of chromic acid solution are allowed to run into the flask through the separatory funnel. This is followed by 50 cc. of sulfuric-phosphoric acid mixture. In case there is water in the sample, 2 cc. of concentrated HzS04 for every cc. of water present are also added. The contents of the flask are mixed by gentle rotation. Heat is applied, very gently at first, and then gradually increased until the solution boils. This usually requires about 20 minutes. If the reaction becomes too violent, as is indicated by the tendency of the reaction mixture to rise in the inlet tube, the flame is removed and heating is resumed only when air again bubbles through the solution. Boiling is continued. until oxida- tion is complete. In the case of difficultly oxidizable substances, such as fats, it may be necessary to add more chromic acid after the solution has boiled for some time.’ In the case of easily ox- idizable substances, such as the sugars, a total digestion periodof 30 minutes suffices. Proteins require a somewhat longer diges- tion period, and it is advisable in the case of substances such as fats to continue the boiling for 1 hour.

The flame is removed, the suction is discontinued, and the re- action flask is disconnected. The stopper at the top of the ab- sorption tower is next removed, and about 200 cc. of carbon dioxide-free water, in 25 to 50 cc. portions, are run in. The flask is removed from the tower, about 25 cc. of M BaClz are added, and the flask is shaken and stoppered. The precipitate of BaC03 becomes granular after a few minutes standing. The residual alkali is then slowly titrated with 0.5 N HCI, with phenol- phthalein as an indicator, to the total disappearance of the pink color. The flask must be shaken or rotated constantly while the acid is being added.

Frequent blanks should be run. The blank corrects for CO2 in the apparatus and reagents, and CO2 absorbed from the at- mosphere during the titration.2 It is unnecessary to use accu-

1 Chromic acid decomposes when heated with strong sulfuric acid. z The blank also corrects for the small amount of acid carried over in

white fumes. According to Kiister and Stallberg (7) the white fumes may be removed by a tube filled with glass wool. More efficient, however, is a tube filled with small glass beads which may be inserted between the reflux condenser and the absorbing tower.

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50 Carbon and Carbon Dioxide

rately standardized NaOH; the same volume, however (measured from an automatic burette) should be present in all determina- tions, including the blank. The titrations may be expressed in terms of the volume of 0.5 N HCl used. The calculation then is made as follows: Volume of HCl used in blank titration - volume of HCl used in titration of sample = HCI equivalent to alkali combined with COZ. 1 cc. of 0.5 N HCl (which must be accu- rately standardized) is equivalent to 3 mg. of carbon.

Determination of Carbon Dioxide.

It is obvious that the apparatus lends itself equally as well for the determination of carbon dioxide as for total carbon. The

TABLE I.

Determination of Carbon Dioxide.

50 cc. of 0.5 N NaOH and 25 cc. of water were used in the tower in all determinations.

Weight of NaKOs.

am. cc. cc.

0.2530 9.52 9.51 0.2530 9.49 9.51 0.2530 9.52 9.51 0.5059 19.02 19.01 0.5059 19.00 19.01

Volume of HCl* equivalent to NaOH

used up.

-

Calculated volume of NaOH used UP. RWW~.

per cent

100.1

99.q 100.0 100.0 100.0

* 1 cc. is equivalent to 0.0266 gm. of Na&08. t The rate of titration with HCI in this case was very rapid.

results shown in Table I were obtained by decomposing pure Na2C03 with HCl. The COZ liberated was aspirated through the towers which contained 50 cc. of 0.5 N NaOH and 25 cc. of water. The recovery of COZ was found to be quantitative only when certain precautions were observed.

The rate of aspiration apparently does not affect the absorption. Complete absorption has been obtained even with a rate of 300 to 400 bubbles of air passing through tube F. The rate of titration, however, may have an influence on the results. If run in very rapidly, the local concentration of HCI may become so great as to decompose some of the BaC03. In consequence of this, more HCI is used and the calculations show a lowered yield.

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T. E. Friedemann and A. I. Kendall 51

Most important, however, is the construction of the tower and the concentration of alkali. Carbon dioxide is rapidly absorbed only by concentrated solutions of alkali. The absorption is very

15 20 25 30

CHART I. Effect of water on the oxidation of acetic acid, alanine, and casein.

markedly influenced by the concentration, and in dilute alkali the absorption is slow and incomplete. A quantitative absorption can be obtained if both the time of contact with liquid and the surface are increased, as can be done, for example, by

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52 Carbon and Carbon Dioxide

aspirating the gas through a tower containing beads. The height of the column of beads in the tower is important. But more important still is the excess of alkali which must be main- tained. With the tower described and the 0.5 N NaOH, an excess

^^I I C’i i i i / i i i i ii i i

cc. cont. l+04

CHART II. Relation between volume of water, volume of H2SOn, and yield of CO2 from the oxidation of acetic acid.

of at least 25 cc. of alkali over that necessary for complete absorption should be present.

Effect of Water and Acid wpon Oxidation.

The effect of water added to the oxidizing reagents is shown in Chart I. Solutions of alanine and acetic acid were measured

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T. E. Friedemann and A. I. Kendall 53

into 300 cc. Kjeldahl flasks, and enough additional water was added to bring the volume to the quantity indicated in the chart. Casein was weighed out, transferred to the flask, and the indi- cated volume of water was added. The oxidation was carried out with 10 cc. of chromic acid solution and 50 cc. of sulfuric-phos- phoric acid mixture. Except in the case of acetic acid, the total

TABLE II.

Recovery of Carbon as Carbon Dioxide by Oxidation of Pure Organic Compounds.

99 to 100 per cent.

Acetic acid. Ethyl alcohol. Crotonic acid. &Hydroxybutyric acid. Lactic acid (lithium salt). Potassium hydrogen saccharate. Sugars.

Sucrose. Glucose. Galactose. Rhamnose.

Methyl amine HCI. Alanine. Aspartic acid. Uric acid. Urea. Dimethylglyoxime. Chinchonine. Potassium hydrogen phthallate. Benzoic acid. Hydroquinone. Piperine.

98 to 99 per cent.

Stearic acid. Carbazole.

time of digestion was 30 minutes. It is apparent that water very markedly affects the yield, presumably by decreasing the rate of oxidation.

The unfavorable effect of water may be overcome by adding concentrated HzS04 to the oxidizing mixture. This is illustrated by the results of oxidation of acetic acid shown in Chart II. 5 cc. of a solution of acetic acid and 20 cc. of water were measured into

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54 Carbon and Carbon Dioxide

Kjeldahl flasks, and the usual amounts of oxidizing reagents, namely 10 cc. of chromic acid solution and 50 cc. of sulfuric-phosphoric acid mixture, were added. 10, 25, 50, and 75 cc. of additional concentrated H&SO4 were added. The total volume of H&SO4 present in the flask therefore was 25 cc. plus the amount of addi- tional acid. The results of 30 and 90 minutes digestion on the yield are shown in Chart II. As can be seen, the unfavorable effect of water can be compensated for by increasing the con- centration of sulfuric acid. 50 cc. of additional H&SO4 in this case were sufficient to bring about complete oxidation even when 25 cc. of water were present.

Oxidation of Pure Organic Substances.

The degree of accuracy of the procedure is illustrated by the analyses shown in Table II. Except for piperine, hydroquinone, and chinchonine, the recovery of carbon from the substances listed in the first column was quantitative or almost quantitative. The yield from stearic acid and carbazole was somewhat low. Both of the substances are very insoluble. In the case of stearic acid it was noted that a very small amount, which could not be dislodged, was always in the condenser at the end of the oxida- tion. Carbazole showed a tendency to creep upward and stick to the side of the flask. In spite of every attempt to wash it down by rotation of the flask during the digestion, a small amount always remained in the upper part of the flask.

SUMMARY AND CONCLUSIONS.

An apparatus and a procedure for the determination of total carbon and carbon dioxide is described. It is pointed out that the wet combustion method is better adapted for materials of biochemical importance than the dry combustion method. The results are quite quantitative, and such difficultly oxidizable sub- stances as acetic acid and fats may be completely oxidized without using catalysts, such as silver or mercury, provided proper at- tention is paid to the total acid concentration.

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T. E. Friedemann and A. I. Kendall

BIBLIOGRAPHY.

1. Rogers, R. E., and Rogers, W. B., Am. J. SC., 6, 352 (1848) ; 6, 110 (1848) ; .I. prakt. Chem., 60,411 (1850).

2. Brunner, C., Ann. Physik, 96,379 (1855); J. prakt. Chem., 67, 11 (1856). 3. Messinger, J., Ber. them. Ges., 21, 2910 (1888). 4. Simon, L. J., Compt. rend. Acad., 179,975 (1924). 5. Florentin, D., Bull. Sot. chim., 36, 228 (1924). 6. Thiele, J., and Morais, J. T., Ann. Chem., 273, 151 (1893). 7. Kiister, F. W., and Stallberg, A., Ann. Chem., 278,214 (1894). 8. Fritsch, P., Ann. Chem., 294,79 (1897). 9. Warrington, R., and Peake, W. A., .I. Chem. Sot., 37,617 (1880). Cross,

C. F., and Bevan, E. J., J. Chem. Sot., 63,889 (1888). 10. Ames, J. W., and Gaither, E. W., J. Ind. and Eng. Chem., 6,561 (1914). 11. Schollenberger, C. J., .I. Znd. and Eng. Chem., 8, 1126 (1916). 12. Kiister, F. W., 2. anorg. Chem., 13, 127 (1897).

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Theodore E. Friedemann and Arthur I. KendallAND CARBON DIOXIDE

THE DETERMINATION OF CARBON

1929, 82:45-55.J. Biol. Chem. 

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