ANALYTICAL CHEMISTRY
948 Accuracy of Method
Table I. Test KO.
Determined, Mg.
Added, Mg.
so2
Total SO2 Found, Mg.
0.105 0.522 0.390 0.625
0.200 0.400 0.100 0.250
0.304 0.930 0.488 0.900
SO?
Table 11. Sulfur Dioxide in Gas Test
Fuchsin Method,
XO,
%
1 2 3
0.27 0.30 0.30 J.29 0.29 0.29 0.30
4 3
6 7
Reich Test.
%
0.28 0.28 0.33 0.35 0.36 0.38 0.45
25" * 3 " C.,for the development of the color. Shake occasionally and measure the per cent light transmitted in the electrophotometer, using a green filter on the spectrophotometer a t 580 mu with the reagent in the reference cell. Analysis of Gas. Scrub the gas through 15 ml. of the sodium hydroxide solution in the apparatus as shown in Figure 1. (The volume of the test tube is small to allow the liquid to rise in the calcium chloride tube to facilitate efficient scrubbing.) The volume of the residual gas collected is dependent on the sulfur dioxide content of the gas. Measure between 50 and 250 ml. of residual gas in the buret by using the leveling bottle. Correct the measured volume to standard conditions (0' C. and 760 mm. of mercury), and adjust for the volume of sulfur dioxide to obtain the total volume of the gas sample. At 0" C. and 760 mm. of mercury, 1 mg. of sulfur dioxide occupies a volume of 0.34. ml. Wash the sodium hydroxide solution into a 500-ml. volumetric flask containing 300 ml. of water, 4.0 ml. of fuchsin solution, and 2.5 ml. of concentrated sulfuric acid (an excess of 100% acid will have no appreciable effect), and proceed as directed for the preparation of the standards. DISCUSSIOV
potassium iodate. Although this method is reliable, there is always danger of losing sulfur dioxide by vaporization in a strong acid solution. The titration is rather tedious for the inexperienced operator, and the end point is difficult to see and can be passed very easily. Haller (5) used normal iodine for titrating the dissolved sulfur dioxide. There is no objection to this method if the sodium hydroxide solution containing the sulfur dioxide is used for titrating an acid solution of normal iodine, and the starch indicator is added just before the end point. This can be observed by the light amber color of the iodine solution. For very low concentrations of sulfur dioxide in a gas, such a procedure would become impracticable. REAGENTS AND APPARATUS
Fuchsin solution is prepared by adding 15 ml. of concentrated sulfuric acid to 200 ml. of distilled water, adding 4.0 ml. of a 3% basic fuchsin solution in alcohol and 1.0 ml. of 40% formaldehyde, and diluting to 250 ml. with distilled water. The sodium hydroxide used is a 10% solution containing 5% glycerol. Standard sodium bisulfite solution is prepared by dissolving C.P. sodium bisulfite in water, so that 1ml. will contain 0.1 mg. of sulfur dioxide. It is standardized by titrating iodometrically. A Fisher electrophotometer or Beckman DU spectrophotometer is used. The absorption apparatus is shown in Figure 1. PROCEDURE
Preparation of Standards. Prepare standards by adding 1 to 10 ml. of the standard sodium bisulfite solutioi. to a 500-ml. volumetric flask containing 300 ml. of water, 4.0 ml. of fuchsin solution, and 2.5 ml. of concentrated sulfuric acid. Dilute to 500 ml. with distilled water and allow to stand 0.5 hour a t room temperature,
For determining the accuracy of the proposed method, known amounts of sulfur dioxide were added to aliquots of samples previously analyzed by the method and the total sulfur dioxide content was determined. Results are shown in Table I. For comparative purposes simultaneous tests of a gas containing 8.0% sulfur trioxide using the above method and the Reich t a t (with sulfuric acid scrubbers) gave the sulfur dioxide content as shown in Table 11. These tests, made a t 5-minute intervals, indicate the stripping of the sulfur dioxide from the concentrated sulfuric acid, giving successively higher results in the Reich test. The sulfur dioxide in the exit stack gas a t the same time was 0.25%, as determined by the Reich test. In the absence of sulfur trioxide, reproducible results can be obtained with the Reich test. Temperature has a definite effect upon the intensity of color produced a t the 0.5-hour period. At 10" C. a concentration up to 2.0 mg. of sulfur trioxide in 500 ml. of solution will also follow Beer's law. Time is also an important factor during the test. Readings should be taken within 5 minutes of the allotted time. A 15minute variation from the 0.5-hour limit will produce approximately a 10% error. LITERATURE CITED
(1) Dragt, G.,and Greenan, K. W., IND.ENG.CHEM:., .~NAL. ELI., 883 (1942). (2) Grant, W.M.,ANAL.CBEM.,19, 345 (1947). (3)Haller, Percy, J. SOC.Chem. Ind.,38,52T (1919). (4)Reis, E.P., and Clark, L. E., Znd. Eng. Chem..18,774 (1926) (5) Steigmsn, A., J. SOC.Chem. Ind.. 61,18 (1942). RECEIVEDJuly 13,1949.
Purification of Ninhydrin by Crystallization PAUL B. HAMILTON A N D PRISCILLA J. ORTIZ The Alfred I . du Pont Institute, Nemoure Foundation, Wilmington, Del.
HE use of ninhydrin (triketohydrindene hydrate) for the Tspecific determination of alpha-amino acids in the ninhydrin-carbon dioxide method (6),for the detection of amino acids in paper chromatography (1), and for the quantitative aolorimetric determination of amino acids in fractions obtained from an i m p o r a t fractionating co~umns(9, 4 ) has established it reagent in amino acid chemistry. Moore and Stein ( 9 ) found a treatmentwith Norit followed by crystallieation from water adequate purification of their ninhydrin. However, a single crystallization from water is not adequate for commercial
ninhydrin that is orange-brown in color, has a strong odor, and contains traces of selenium. The following procedure has been found efficient and gives a good product in high yield with few crystallizations. grams) is dissolved in 500 ml. of hot Cntde ninhydrin 2 N hydrochloric acid and an brown scum is removed with a spatula. Acid-washed neutral &orit (10 grams) is added, and the solution is allowed to boil gently for about 10 minutes, and then filtered hot through a medium-pore fritted disk. Hot saturated solutions of ninhydrin are somewhat orange in color, but when cool are very pale yellow. Crystallization of ninhydrin from the
V O L U M E 22, NO. ?, J U L Y 1 9 5 0 filtrate is allowed t o take place slowly with occasional stirring while the solution cools to room temperature. Crystallization is completed a t 4' C. for 16 hours. The crystals are collected on a fritted disk, washed three times with 50-ml. portions of ice-cold 1N hydrochloric acid, and sucked free of excess liquid. Residual water and hydrochloric acid are removed in 24 hours in an evacuated desiccator over solid potassium hydroxide. The yield is approximately 94% of the starting material. When dry, the product is stored in an amber bottle.
60 mm. gas pressure, measured a t the 0.5-ml. marked in the Van Slyke-Neil1 manometric apparatus ( 7 ) , in excess of the normal blank analysis in the ninhydrin carbon dioxide method (6). After purification, two treatments with Korit, each followed by crystallization, a product was obtained with properties as described above and which no longer contributed to the gas pressure of the blank ninhydrin carbon dioxide analysis.
Pure ninhydrin is very pale yellow with a greenish tint, is completely odorless, and dissolves in water to give a clear light yellow solution. Ninhydrin purified as above turns pink a t 125' C., shrinks with loss of water of hydration, becomes deep purple red at 139-140" C., and melts sharply with decomposition a t 241 ' C. (uncorrected). Corrected for stem temperature the melting point is 247" C. Ruhemann (8)reported similar observations and found a melting point of 239-240' C. (uncorrected). Teeters and Shriner ( 5 )recorded a value of 241-243 ' C. but did not state in their publication whether this was a corrected melting point. Samples of ninhydrin have been found which contribute 50 to
( 1 ) Consden, R., Gordon, A. H., and Martin, A. J. P., Biochem. J . , 38,224 (1944). (2) Moore, S., and Stein, W. H., J . B i d . Chem., 176, 367 (1948). (3) Ruhemann, S.,J. Chem. Soc., 97,1446 (1910). (4) Stein, W. H., and Moore, S., J . Biol. Chem., 176,337 (1948). (5) Teeters, W. O., and Shriner, R. L., J. Am. Chem. Soc., 55, 3026 (1933). (6) Van'Slyke, D. D., Dillon, R. T., MacFadyen, D. A., and Hamilton, P. B., J . Biol. Chem., 141, 627 (1941). (7) Van Slyke, D. D., and Neill, J. M., Ibid., 61, 523 (1924).
LITERATURE CITED
RECEIVED September 0, 1949.
Kjeldahl Microdetermination ROBERT M. SILVERSTEIN A N D ROBERT PERTHEL, JR. Stanford Reseerch Institute, Stanford, Calif. HREE improvements with respect to accuracy and conTvenienoe in the micro-Kjeldahl procedure have been made. The first modification is designed to eliminate the occasional, but extremely annoying, suck-back of material into the steam generator, or of distillate and receiving aoid back into the distillation flask. This is accomplished by means of a simple, allglass check valve on the steam generator.
type in which the steam generator surrounds the distillation flask; there are numerous occasions when interruption without suok-back of a steam distillation in a Pozzi-Escot a paratus (7) may be desirable. At the completion of a KjeldahPdistil+ tion, the flame is removed, and the distillation flask emptied u1 the usual way by holding a fingertip over the tip of the valve.
In Figure 1, the valve is shown on a Kirk-type Kjeldahl apparatus ( 8 ) . It consists of a solid,.ground-glass ball, a ground ring seat 1 mm. in width, and four mdentations placed to allo: approximately a Zmm. lift. The seat is ground in with a 45 taper brass grinding tool. This valve is applicable to steam distillation setups in general, and is particularly effective on the n
M
,-moo 0
r7mm00
The practice in this laboratory had been to immerse the delivery tip (8 mm. in outaide diameter) of a Kirk apparatus in 5 ml. of 2% boric acid in a tilted 50-ml. Erlenmeyer flask. Distillation had been carried out with the tip immersed for 3 minutes, and then for 1 minute with the tip above the liquid ( 5 ) . The slightly low results obtained with pure ammonium sulfate samples indicated incomplete absorption of ammonia. Therefore, in order to facilitate absor tion of ammonia and still use the smallest possible amount of goric acid, a small bulb was blown on the delivery tip and five holes were unched therein with a hot, 1-mm. tungsten wire (Figure 2). !his bulb was immersed in 5 ml. of boric acid in a 20 X 150 mm. test tube. The use of the test tube provided a greater depth of boric acid, and permitted distillation of a definite volume (appropriate marks on the test tube) rather than the indefinite amount which varied with the rate of distillation during the fixed time interval. Eight milliliters were collected with the ti immersed and 2 ml. with the t i raised. A quantitative transfer to a suitable titration flasE with two 1-ml. washings brought the total volume t o about 17 ml. Figure 2
U
Figure 1 Diagram of Check Valve
An investigation carried out in this laboratory into the cause of frequent, slightly low results obtained on standard samples led to the second modification of the Kirk apparatus. Willits, John, and Ross (8) obtained "slightly low nitrogen values" on pure nitrogeneous compounds, using the Kjeldahl macroprocedure of the kssociation of Official Agricultural Chemists. These results were accounted for by assuming that all the ammonia was not caught by the trapping liquid. Subsequent use of a Goessman trap ( 1 ) on the receiver gave higher and more nearly theoretical results.
