1332
ANALYTICAL CHEMISTRY
unknown solution. In practice, amino acids which are most likely to be off the calibration curves (glutamic acid, glycine, methionine, etc. ) are readily estimated on one-dimensional chromatograms. After an approximate idea of the composition of the unknown solution has been attained by either of the above methods, a mixture of standard amino acids is prepared which contains the same molar concentration of each substance as is estimated to be prese n t in the unknown solution. A number of replicate chromatograms are then carried out simultaneously on this standard mix. ture and on the unknown. The results of two separate experiments based upon the color ratio method and on calibration curves, respectively, are given in Table 111, together with earlier results (2) obtained from many more chromatograms using the color ratio method described above. A similar proportionality between color density and concentration has been found on chromatograms of histamine, cadaverine, putrescine, tyramine, phenylethylamine, propylamine, isobutylamine, and n-amylamine. The amines were separated from amino acids and peptides by extraction with ether in a continuous extractor from a dilute alkaline solution.
LITERATURE CITED (1)
.Acher, R., Fromageot, C., and Jutisz, M., B i o c h a . el Biophys.
(2) (3) (4)
Block, R. J., Proc. SOC.Erptl. B i d . Med., 72, 337 (1949). Block, R. J., Science, 108, 608 (1948). Block, R. J., and Bolling, D., “Amino Acid Composition of Proteins and Foods,” 2nd ed., Springfield, Ill., C. C Thomas,
(5)
Bull, H. B., Hahn, J. W., and Baptist, V . H., J . Am. Chem. SOC.,
(6) (7)
Bush, M. T., and Densen, P. M., ANAL.CHEM.,20, 121 (1948). Consden, R., Gordon, A. H., and Martin, A. J. P., Biochem. J.,
Acta, 5, 81 (1950).
1950. 71, 551 (1949).
38,224 (1944).
Dent, C. E., Ibid., 43, 169 (1948). (9) Fisher, R. B., Parsons, D. S., and Holmes, R., Nature, (8)
(10)
Fisher, R. B.. Parsons, D. S., and Morrison, S. A., Ibid.,
161,764
(1948). (11)
Martin, A. J. P., Ann. Repls. on Progress C h a . (Chem. SOC.
(12) (13)
Polson, A,, Biochim. et Biophys. Acta, 2. 575 (1948). Rockland, L. B., andDunn, M. S., J . Am. Chem. Soc.,
London), 45,267 (1949). 71, 4121
(1949).
(15)
Williams, R. T., and Synge, R. L. M., “Partition Chromatography,” Biochemical Society Symposia No. 3, Cambridge, Cambridge University Press, 1949. Williamson, B., and Craig, L. C., J . Biol. Chem., 168, 687
(16)
Winegard, H. M., Toennies, G.,.and Block, R. J., Science,
(14)
(1947).
ACKNOWLEDGMENT
The author is indebted to Herbert A. Sober for assistance in this study and to David Miller for the statistical evaluations. This investigation w m aided in part by a grant from The Borden Company, New York 17, N. Y.
164, 183
(1949).
108,
506 (1948). (17) (18)
Woiwod. A. J.. Biochem. J.. 45, 412 Work, E., Lancet, 256, 652 (1949).
(1949).
RECEIVED January 31,1950.
Microdetermination of Sulfur by the Grote Method Photometric Detection of the Titrimetric End Point ROLAND N. WALTER Hercules Experiment Station, Hercules Powder Company, Wilmington 99, Del.
In the Grote combustion micromethod for determining sulfur in organic compounds, the sulfate formed is usually titrated with standard barium chloride solution, using tetrahydroxyquinone (THQ) or an alkali salt of rhodizonio acid as an internal indicator. A photometric method of detectirig the end point, which is more objective than the usual visual method, is described. Titration results are reproducible within 0.004 mg. of sulfur.
F
OR the determination of sulfur in small samples of organic compounds, a microprocedure must be used. The recent literature on this subject has been well summarized by Willits (6),who, with Ogg and Cooper (X),described a visual titrimetric technique for the identification of the end point in the volumetric determination of soluble sulfates with barium chloride and dipotassium rhodizonate. Hallett and Kuipers ( 1 ) had previously used a somewhat similar technique. Steyermark, Bass,and Littman ( 4 ) have applied the titrimetric technique of Ogg et al. (2) t o the analysis of organic compounds decomposed by the Carius method. A study of the transmittance curves of disodium rhodizonate and of tetrahydroxyquinone (THQ) at the end point showed that the change in color a t this point is small. Accordingly, in order to have a more objective method of identifying the end point, and because of the experience of others in this laboratory with the use of photoelectric photometers (8)in detecting the exact end points of titrations, the writer has made use of these instruments in the present work.
The spectral characteristics of tetrahydroxyquinone and disodium rhodizonate indicators were obtained by means of a General Electric recording spectrophotometer. Figures 1 and 2 show transmittance curves with potassium sulfate and indicator, and curves with potassium sulfate, indicator, and a 5% excem of 0.02 N barium chloride. The two sets of curves are very similar. Because a t the end point a change in hue takes place, the wave length a t which the greatest change in transmittance occurs is not necessarily a t the absorption maximum (480 mp). Actually it occurs a t a point between 520 and 530 mp. At the absorption maximum, the transmittance increases with addition of barium chloride, while it decreases a t wave lengths above 500 mp. In the early experimental work, a specially designed Hercules general-purpose photometer and a tristimulus blue filter (Henry A. Gardner Laboratory) with a transmittance maximum a t 440 mp were used. Later a KletbSummerson photoelectric colorimeter and a Baird interference filter having a transmittance maximum a t 537 mp were found to be equally suitable. With the latter combination it was possible to identify the end point by
V O L U M E 2 2 , NO. 10, O C T O B E R 1950 the deflection of the galvanometer needle, thus eliminating the necessity of plotting a curve. This new technique is advantageous when the method is used routinely. APPARATUS
Klett-Summerson photoelectric colorimeter. Green interference filter with a transmittance maximum a t 520 to 540 mp. Titration cell (Figure 3). A threehole No. 6 rubber stopper used t o close the titration cell and to hold the stirrer shaft and buret tlp.
loo
Figure 1.
1333 saturated bromine water are added to oxidize any sulfite to sulfate. An amount of 0.1 N nitric acid slightly greater than the volume of 0.1 N sodium hydroxide used a t fist is then added. The solution is boiled until the excess bromine is dispelled and the volume is reduced to about 15 ml. After the solution has been cooled and treated with 2 drops of 1% phenolphthalein solution, it is made slightly alkaline with 0.1 N sodium hydroxide and then faintly acid with 0.01 N nitric acid. This solution is transferred to the photometer titration cell, and an equal volume of alcohol and about 0.15 gram of indicator are added. The green interference filter is placed in the Klett-Summerson photoelectric colorimeter, the titration cell is inserted, and the cell is closed with the three-hole No. 6 rubber sto per which has the mechanical stirrer shaft extending through t i e center hole. The height of the stirrer is adjusted so that the rubber blades fall in the center of the bulb portion of the cell. The stirrer is run a t a moderate speed. The offset buret tip is inserted through one of the remaining holes until its end is below the surface of the solution. Next, the density dial of the photometer is set a t zero and the galvanometer adjusted to zero current by means of the null control which is connected to the balancing photocell. The barium chloride solution is now added a t a rate of about 3 ml. per minute. When about two thirds of the required amount has been added, there will be a considerable deflection of the galvanometer needle. This is caused by the temporary formation of the red-colored barium salt of the indicator, which decreases the transmission of the solution. When the needle has been deflected a distance corresponding to 30 or 40 dial units, the addition of barium chloride is stopped for 5 to 10 seconds, t o allow the pseudo end point to disappear. As soon as the needle returns to a stable deflection (not necessarily zero), the addition of titrant is resumed in 0.05ml. ortions. In order to keep the galvanometer needle on the scaE, it is necessary t o turn the density dial slightly from time to time; this should be done after the needle has come to a stable position.
Spectral Tranemittance Curves for THQ Indicator (Betz)
A mechanical stirrer consisting of a small electric motor and shaft. As a propeller, diagonal grooves are cut in the lower half of a No, 5 rubber stopper. A 5-ml. buret graduated in 0.01-ml. divisions, with interchangeable tip. For flexibility in operation, an auxiliary buret tip is inserted through the rubber stopper and connected to the buret by means of a binch piece of Tygon tubing. REAGENTS
Potassium sulfate solution, 0.02 N , prepared from dried A.C.S.grade material. Barium chloride solution, 0.W N , prepared and standardized against the 0.02 N potassium sulfate solution, using the procedure described subsequently for the titration of samples. Nitric acid solution, approximately 0.1 N . Sodium hydroxide solution, approximately’ 0.1 N . Indicator. The prepared THQ indicator sold by Bet2 Laboratories, Philadelphia, Pa., is already diluted with an mert material. The pure disodium rhodizonate sold by Smith-New York, Inc., Freeport Long Island, N. Y,, must be diluted 1 to 300 with powdered sucrose. PROCEDURE
The combustion of the organic sample is carried out according to the method of Sundberg and Royer (6). The solution of combustion products in 0.1 N sodium hydroxide is washed from the Grote absorber and vapor trap with a minimum amount of distilled water into a 125-ml. Erlenmeyer flask, and 5 ml. of
Figure 2. Spectral Transmittance Curves for Disodium Rhodizonate Indicator (Smith-New York)
When the end point is imminent, the concentration of the solution in terms of sulfate has become small, and the time required to dispel the pseudo end point becomes longer. As soon as the galvanometer needle becomes sluggish in its return to a stable position, the barium chloride additions are decreased to 0.02ml. portions.
A N A L Y T I C A L CHEMISTRY
1334
ment of the needle is noted after its deflection to the right. In accordance with the procedure
7 2 5 MY.
tion are required. The reason for this step is that the indicator apparently does not have enough barium sulfate surface upon whieh to absorb unless this much barium chloride is used.
SUMMARY
85 YW.
DI SCU 9 SION
K
In the early work on the titration
Table I.
small quantities of potassium sulfate are shown in Table I and indicate an accuracy and precision of a t least 0.004mg. of sulfur. Table I1 contains the results found on determining sulfur in' four compounds by the foregoing procedure. In this work both the Hercules general-purpose photometer and the Klet,t-Summerson photoelectric colorimeter were used. The Parr bomb results were obtained gravimetrically.
A photometric method of detecting the end point in the titration of the sulfate formed in the microdetermination of sulfur by the Grote method is described. The proposed method does not require the construction of a curve. It is less subjective than the usual visual method and should assist in overcoming objections to the use of an internal indicator such as tetrahydroxyquinone.
22".
Titration of Known Amounts of Potassium Sulfate
(Using Klett-Summerson photometer and green filter) S Present, Mg. S Found, Mg. 1.234 1.233 1.234 1.230 1.234 1.234 1.234 1.233 1.851 1.847 1.851 1.850 1.851 1 ,850 3.085 3.082 YL. OF 0.02 C B 4 R I U Y C H L O R I D E
Additional tests were made with the Klett-Summerson photometer, substituting a green interference filter for the blue filter. This new combination proved to be sufficiently sensitive for detection of the end point by observing the deflection of the galvanometer needle, as before. The results of titrations of known
Table 11. Comparison of Methods for Determination of Sulfur in Known Compounds
Cystine
Herculea generalpurpose photometer 26.69 26.73 26.75 26.99
Sulfur Found, % KlettSummerson photoelectric colorimeter 26.82
2,4-DichlorophenyI-4toluene sulfonate
10.09 10.35
...
Sulfonal
27.95 28.21
...
S-Benzylthiuronium chloride
...
15.86 15.77
ACKNOWLEDGMENT
The author wishes to thank Robert H. Osborn for helpful suggestions during the course of this work. LITERATURE CITED
% ,
26.69
10.00 10.20 10.28 10.28
10.11
28.4 28.2 28.6
28.09
...
This technique should be applicable when other makes of photoelectrib photometers or spectrophotometers are used, and with any method for determining sulfur in which the sulfur is oxidized to sulfate, such as in the Carius tube, peroxide bomb, or oxygen bomb.
Sulfur Calculated,
Parr bomb
...
Pigure 4. Titration Curve Showing End Point Using Klett-Summerson Photometer with Blue Filter
15.82
(1) Hallett, L. T., and Kuipers, J. W., I&. ENO.CHEM.,ANAL.ED., 12, 360 (1940). (2) Om,C. L., Willits, C. O., and Cooper, F. G., ANAL.CHEM.,20, 83 (1948). (3) Osborn, R. H., Elliott, J. H., and Martin, A. F., IND. ENG. CHEM.,ANAL.ED., 15, 642 (1943). (4) Steyermark, Al,Bass, E., and Littman, B., ANAL.CHEM., 20, 587 - - - (1948). (5) Sundberg, 0. E., and Royer, G . L., IND.ENCI.CHBIM., ANAL.ED., 18, 719 (1946). (6) Willits, C. O., ANAL.CHEY., 21, 136 (1949).
.----,
RECEIVED December 10, 1949. Presented at the Symposium of the Delaware Section, AMERICAN CHEMICAL SOCIETY, Newark, Del., January 14, 1950.
