762
INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 14, No. 9
Discussion
Acknowledgment
The mechanism of the apparent loss of nitrogen is not clear. Mears and Hussey suggested that possibly an excess of perchloric acid formed ammonium perchlorate which then decomposed. Walters, Smith, Willard and Cake, and Peters and Van Slyke evidently believed that part of the ammonia (as ammonium sulfate) could be oxidized to free nitrogen. Myers regarded the lower results in colorimetric micro determinations as probably due to the formation of amines which, he stated, would not give full color development with Nessler’s reagent. This does not seem to be sufficient explanation, although Gortner and Hoffman (9) pointed out that about 7 per cent of amine nitrogen results with ordinary Kjeldahl digestion, and Villiers and Moreau-Talon (20)earlier claimed that the presence of strong oxidizers promotes amine formation. (Their paper was prior to the introduction of perchloric acid.) Certain analysts may protest that, granting all the above, if an excess of perchloric acid be avoided, the error will still be negligible. This may be true for sizable samples, and where just enough perchloric acid is added to clear the solution after preliminary digestion by sulfuric acid. But for microquantities, even a small excess may release a considerable portion of the nitrogen-for example, 2 small drops may be inadequate and 3 drops excessive.
The authors are grateful to G. F. Smith of the University of Illinois for the loan of a monograph (8) otherwise difficult to obtain. Literature Cited
Temperature Apparently temperature has an important influence on the oxidizing power of perchloric acid. The acid does not seem to have much effect on the organic matter until sufficient water has been driven off from the digest to elevate the boiling point considerably, after which the combustion is vigorous and rapid. A little perchloric acid added then is much more potent than a great deal present in the original digestion mixture. Certain experiments suggest that, with very careful temperature regulation, a zone might be found where one could complete the oxidation of the carbon without loss of nitrogen. The authors wonder, however, whether such procedure would prove either practical or trustworthy.
(1) Chiles, H. M., J . Am. Chem. Soc., 50, 217 (1928). (2) Doneen, L. D., Piant Physiol., 7, 717 (1932). (3) Dupray, M., J . Lab. Clin. Med., 12, 386 (1926).
(4) Fisaerman, J. H., and Fiszerman-Garber, D., Bull. SOC.Pharmacol., 40, 210 (1933). (5) Folin, O., and Wu, H., J . B i d . Chem.. 38, 81 (1919). (6) Frey, R. W., Jenkins, L. J., and Joslin, A. ~ .J . ,Am. Leather Chem. Assoc., 23, 397 (1928). (7) Fuchs, H. J., and Falkenhausen, hl. Y., Biochem. Z., 245, 304 (1932). (8) Gauduchon-Truchot, H., “Contribution 1 l’Etude de la MMBthode de Kjeldahl”, Paris, Imprimerie Henry, 1936. (9) Gortner, R. A., and Hoffman, IT. F., J . B i d . Chem., 70, 457 (1926). (10) Kito, W. H., A n a l y s t , 59, 733 (1934). (11) Koch, F. C., and McMeekin, T. L., J . Am. Chem. Soc., 46, 2066 (1924). (12) Lematte, L., Boinot, G., and Kahane, E., J . pharm. chim., 5, 325, 361 (1927). (13) LeTourneur-Hugon and Chambionnot, Ann. f a l s . , 29, 227 (1936). (14) Mears, B., and Hussey, R., J. ISD. ESG. C H m r . , 13,1054 (1921). (15) Myers, V. C., J . Lab. Clin. Med., 17, 227 (1931). (16) Parker, J. G., and Terrell, J. T., J . SOC.Leather Trades Chem., 5, 380 (1921). (17) Peters, J. P., and Van Slyke, D. D., “Quantitative Clinical Chemistry. Methods”, p. 519, Baltimore, \Tilliams & Wilkins Co., 1932. (18) Rose, A. R., J . B i d . Chem., 64, 253 (1925). (19) Smith, G. F., IND.ENG.CHEY.,ANAL.ED.,6, 229 (1934). (20) Villiers, A., and Moreau-Talon, A , , Bull. soc. chim., 23, 308 ( 1918). (21) Walters, L. C., -4ustralian J . Erpcl. BioZ. M e d . Sci., 7 , 113 (1930). i i ~ aED., ~ . 4, (22) West, E. S., and Brandon, A . L., IND.E N G .CHEM., 314 (1932). (23) Wicks, L. F., J . Lab. Clin. M e d . , 27, 118 (1941). (24) Willard, H . H., and Cake, W. E., J . Am. Chem. Soc.. 42, 2646 (1920). (25) Wong, S. Y., J . Bid. Chem., 55, 431 (1923). (26) Yoe, J. H., Ann. chim. anal., 7, 193 (1925). (27) Zakrzewski, Z., and Fuchs, H. J., Biochem. Z., 285, 391 (1936).
Improvements in the Colorimetric Microdetermination of Phosphorus C. P. SIDERIS, Pineapple Research Institute of Hawaii, Honolulu, T. H.
T
W O fundamentally distinct methods have been in use for
almost two decades for the colorimetric determination of phosphorus-the Bell-Doisy (1) and Copaux ( 4 ) methods. The former, depending on the estimation of a blue color formed by the yellow phosphomolybdate compound reacting with some reducing agent, has undergone many modifications in the hands of various workers. A very excellent account of this method and modifications has been presented by Snell (6) and Peters and Van Slyke ( 5 ) . Zinzadze’s studies (7, 8 ) pertaining to amounts of reducing reagent, time, temperature, etc., are interesting. A great improvement in the colorimetric determination of phosphorus was made with the introduction of the Berenblum-Chain (2, 3) method. Incorporating the features of both the Bell-Doisy and Copaux methods, this proved more successful than either. In the Berenblum-Chain method the yellow phosphomolybdate compound is extracted with isobutyl alcohol instead of ether as in the method of Copaux, and then changed into the blue phosphomolybdate compound by
means of a reducing agent (stannous chloride in 37 per cent hydrochloric acid). Also, the ether phosphomolybdate extract obtained by the method of Copaux can be easily converted into a blue color by addition of the tin-hydrochloric acid reagent used by the writer. However, this procedure is not recommended, owing to the high degree of volatility of the ether and rapid changes in the volume of the extract. nButyl alcohol is preferred to ether as a solvent. The main points of divergence of the writer’s technique from that of Berenblum-Chain are in the use of metallic tin instead of stannous chloride in 37 per cent hydrochloric acid, elimination of ethyl alcohol for washing the separatory funnel and dilution of the blue phosphomolybdate compound, heating the mixture containing the unknown, 2 N sulfuric acid, and ammonium molybdate, and the use of normal instead of isobutyl alcohol. A reducing agent prepared by mixing stannous chloride and 3 i per cent hydrochloric acid sometimes loses its reducing power and fails to give an intense indigo blue color during an
ANALYTICAL EDITION
September IS, 1942
TABLEI. READINGSON SUMMERSON-KLETT PHOTOELECTRIC COLORIMETER Phosphorus .Mg./l.
Scale Readingso
Scale Readingsb
yo Loss of Colorb
Scale Readingso
5% of Original Colore
1.5 .. .. 0 26 47 0.5 50 48 50 68 32 1 .o 74 90 64 36 1.5 100 15 110 127 130 2.0 130 150 2.5 13 ~~. 146 175 152 180 3.0 15 200 171 22 220 3.5 234 250 206 4.0 17 265 223 4.5 20 280 300 245 23 320 5 0 340 302 19 370 6.0 440 420 370 7 0 16 8 0 470 420 11 455 9 0 540 480 11 530 a 2 hours after extraction. b 72 hours after extraction. C After addition of tin-HC1 reagent to 72-hour standing samples.
..
94 92 90 98 97 97 91 94 95 94 92 96 97 98
entire day's work and often the color of samples has a greenish blue tinge. The metallic tin-hydrochloric acid reducing agent, as recommended by the writer, is considerably more effective than the stannous chloride-hydrochloric acid reagent of Berenblum-Chain, so that reduced samples seldom possess a greenish blue tinge. The use of n-butyl alcohol instead of ethyl alcohol retains the water-immiscible properties of the phosphomolybdate extract, a n advantage for samples which have failed to undergo complete reduction to the indigo blue color so desirable for an accurate determination of phosphorus. Such samples can be treated again, a t any time, with the metallic tin-hydrochloric acid reagent in a separatory funnel until the indigo blue color is obtained, without appreciable loss of the phosphomolybdate extract. The choice of n-butyl alcohol over isobutyl alcohol is of no great importance and either form of butyl alcohol will serve the purpose very well.
Reagents TIK-HYDHOC~HLORIC ilc10. Place 0.5 gram of metallic tin (mossy tin) in a 50-ml. volumetric flask containing 40 ml. of 37.5 per cent hydrochloric acid, and allow to stand for 1or 2 hours, agitating the mixture gently at intervals until complete solution. AMMONIUM MOLYBDATE.Dissolve 40 grams of ammonium molybdate in 1000 ml. of water and filter. n-BuTYL ALCOHOL. SULFURIC ACID,5.6 PERCENT. Prepare a 5.6 per cent (2 N ) solution of sulfuric acid by pouring 56 ml. of concentrated sulfuric acid (density 1.84) into a 1000-ml. volumetric flask containing 500 ml. of water. Cool and complete the volume with water. SULFURIC ACID(1.4 PERCEKT0.5 N ) A N D TIX. Transfer 50 ml. of the 5.6 per cent solution of sulfuric acid to a 200-ml. flask, add 10 ml. of the tin-hydrochloric acid reagent, and make to the mark with water.
763
of the two layers. The emulsion can be easily broken up by the addition of 5 ml. or more of 0.5 N sulfuric acid. Discard the aqueous layer in all cases; add 10 ml. of the reagent of tin-hydrochloric acid in 0.5 N sulfuric acid to the butyl alcohol layer containing the yellow phosphomolybdate compound, and agitate the mixture vigorously. After allowing sufficient time for the separation of the two layers, discard the lower aqueous layer. If the phosphomolybdate compound shows a greenish blue tinge, add more of the tin-hydrochloric acid reagent until the blue color is entirely free from a greenish tinge. After the removal of the aqueous layer transfer the blue butyl alcohol layer to a 15-ml. or any other convenient size graduated centrifuge tube, and, if necessary, add more butyl alcohol to the separatory funnel to wash away all traces of the blue phosphomolybdate compound which is added to the extract in the centrifuge tube. Finally adjust the volume of the butyl alcohol-phosphomolybdate extract in the centrifuge tube by the addition of more butyl alcohol. The addition of 2 to 5 ml. of the tin-hydrochloric acid reagent to the butyl alcohol extract in the centrifuge tube was found to stnbilize and retard the fading of the blue color. However, the addition of the tin-hydrochloric acid reagent often causes a slight emulsification of the extract which can be easily broken up by centrifugation in the course of 5 to 10 minutes. After centrifugation leave the tin-hydrochloric acid in the tube to occupy the loner layer while a volume of approximately 8 ml. of the butyl alrohol layer containing the phosphomolybdate compound is transferred by means of a pipet to a glass cell or a tube and phosphorus is determined by means of a visual colorimeter, a photoelectric colorimeter, or a step photometer with appropriate light filt ws.
Experimental Results The data reported in Table I were obtained by the above method with known amounts of phosphorus. The scale readings for known amounts of phosphorus were obtained with a Summerson-Klett photoelectric colorimeter employing a KO. 59 light filter with transmission limits of 565 to 630 millimicrons and a 2.5-mm. cell. The 2.5-mm. thickness of the cell was obtained by inserting a 7.5-mm. thick plunger into a 10-mm. cell. Three sets of readings are presented in Table I, obtained (1) 2 hours after the extraction of the butyl alcoholphosphomolybdate compound; (2) after 72 hours of standing in ordinary light and a t room temperature (25" to 29" C.); and (3) after the addition of 5 ml. of the tin-hydrochloric acid reagent to the samples which had stood 72 hours.
Method of Procedure Ash 1 or 2 grams of dry plant tissue in a platinum dish, dissolve the ash with 10 ml. of 5 N hydrochloric acid, then add 20 ml. of lvater and heat on a hot plate (90" C.). Pour the solution into a 100-ml. volumetric flask, add water to complete the volume, and filter. Transfer an aliquot of 10 ml. of the filtrate into a Pyrex ignition tube (25 X 200 mm.), neutralize with 5 N sodium hydroxide, using a small piece of litmus paper as indicator, then add 2 ml. of 2 N sulfuric acid and 5 ml. of ammonium molybdate, and heat in a water bath for 10 minutes. (The heating of the mixture was found to facilitate the extraction with butyl alcohol, besides speeding the formation of the phosphomolybdate compound.) Pour the mixture into a 250-ml. Squibb separatory funnel, rinse the tube with 5 ml. of hot 0.5 N sulfuric acid, and transfer the washings to the funnel. Add 10 ml. of n-butyl alcohol, close with a glass stopper, and agitate vigorously for 30 seconds. upper yellowish phosphomolybdate layer to separate from he h e r aqueous acidified layer. Often an emulsion forms between butyl alcohol and \rater, requiring a long time for the separation
Pe
d
I
1
I
2
MG.OF
I
3
I
4
PHOSPHORUS
I
5
I
6
1
7
I
8
PER LITER OF SOLUTION
FIGURE1. DETERMINATION OF PHOSPHORUS
I
9
1
Vol. 14, No. 9
INDUSTRIAL AND ENGINEERING CHEMISTRY
764
Literature Cited
The reagents showed a color reading of 15 scale units, representing a phosphorus contamination of approximateb 0.2 microgram of phosphorus per ml. of solution. The culve drawn in Figure 1 was set for its zero phosphorus value at 15 of the colorimeter scale, because of the phosphorus contamination of the reagents, and for its 9 micrograms of phosphorus value a t 525 (540 - 15 = 525). The deviation of the various experimental points ranges from 0 t o 7 per cent. The writer wishes to warn against the use of greater coneentrations of sulfuric acid than those recommended and the adding of the tin-hydrochloric acid reagent to hot butyl alcohol extracts. Check always with one or more standard solutions the values .. of.unknown samples . . if obtained . . . from curves based on readings irom a photoelectric colorimeter scale
(1) B ~ I IR. , D.. and ~ ~E. A.,iJ . ~ i o~ z Chm., . ~ 44,55 , (1920). (2) Berenblum, Isaac, and Chain, Emst, Biochm. J., 32. 287-94
!:i
(1938). ~
o
$
;
(5) Peters, J. p., and
~
~ 173, ~ 656~(1g21). ~
~
~
~
~
d
vanslyke, D. D.. “Quantitative clinical Chem-
istry. Vol. 11, Methods”. Baltimore, Willimns &- Wilkins Co., 1932. (6) Snell, F. D.. and T. C.. “Colorimetric Methods of Analysis”, Vol. I, New York, D. Van Nostrand Co., 1936. (7) Zineadee, s. R,, z, P3amenernBhv, Diiwung Bdmk,, 12984 (1930). (8) Ibid.. 23, 447-54 (1932). with the appTovaiof the acting director a Technical paperN ~ . 140 of the Pineapple Researoh Institute of Hawaii. University of Hawaii.
PUBL,BBED
Gas-Fired Furnace for ! emimicrodetermination of Carbon a id Hydrogen U ARMIN PACRT.. 4u-m
T.ahnratnvv nf Cha
&try, University of Nebraska, Linooln, Nebr.
T
HE successful combustion of the sample is a very impor-
tant factor m the determination of carbon and hydrogen, The author has long felt that certain more or less serious diffieulties encountered in the combustion operation could be reduced considerably. Among these difficulties are (1) the combustion of graphitic carbon residues, (2) the uncertainty of comdete oxidation of the oreanic vanor. and (3) mevention
FIGURE 1. ASSEMBI
’
When the conventional electric furnace type of apparatus is used it becomes necessary to slide the furnace over the carhon residue in order to complete the combustion. Because of the creeping tendency of many types of organic compounds, carbon residues are particularly apt to be deposited on the upper side of the bore of the combustion tube for a considerable distance. esuecially when large samples are used. It
ridation of organic if they pass too
,
