April 15, 1932
IN DU STR IA L A N D EN G INE ER IN G C HE M ISTRY
(13) Ziritl and Rienacker, 2. anorg. allgem. Chem., 161, 374, 385 (1927); Brintzinger and Oschats, Ibid., 165, 221 (1927); Brintzinger and Rodis, Ibid.,166, 53 (1927) and 2. Elektroehem., 34, 246 (1928); Zintl and Zaimis, 2. angew. Chem., 40, 1286 (1927) and 41,543 (1928); Zintl, Rienacker, and Schlof-
243
fer, 2. anorg. allgem. Chem., 168,97 (1927) ; Zintl and Schloffer 2. angew. Chem., 41, 956 (1928); Brintzinger and Schieferdecker, 2. anal. Chem., 76, 277 (1929) and 78, 110 (1929). RECEIVED June 9, 1931.
Microdetermination of Protein in Cereal Products REXJ. ROBINSON AND J. A. SHELLENBERGER, University of Washington, Seattle, Wash. ICROMETHODS of analysis were originally developed for use when only a small sample of the material could be obtained. More recently they have proved so accurate, rapid, and economical that they are now used in many determinations even when there is an abundance of sample available. This paper deals with an adaptation of the microdetermination of nitrogen for usage in the cereal industries, just as Bermann (3) applied micromethods to the determination of nitrogen in the fermentation industry.
PROCEDURE Using a microchemical balance, from 10 to 20 mg. of sample are weighed into a micro-Kjeldahl digestion tube. A small crystal of copper sulfate and 2 ml. of concentrated nitrogenfree sulfuric acid are then added. The sample is thoroughly mixed with the acid and then heated until the organic matter is charred. This usually requires about 4 minutes. A microKjeldahl digestion rack is very convenient for use in this operation, since several samples may be digested a t the same time. The digestion mixture is allowed to cool, after which 1 gram of potassium persulfate is added ( 7 ) . The mixture must be cooled to at least 100’ C., otherwise the potassium persulfate decomposes a t the surface of the hot liquid, producing an inefficient oxidation. The mixture is again heated gently until complete decomposition of the potassium persulfate has taken place. A perfectly clear sample is usually obtained after heating approximately 1 minute, and the time for the entire digestion is usually less than 10 minutes. A distillation equipment similar in principle to that of Kemmerer and Ballett (4) was used for this investigation and gave accurate results, although it was not so suitable for routine work as their more elaborate apparatus. The digested sample is washed into the distillation flask and sufficient 40 per cent carbonate-free sodium hydroxide solution added to neutralize the excess sulfuric acid. The ammonia is expelled by steam distillation, assisted by the heat from a microburner beneath the distillation flask. About 25 ml. of the distillate are collected in a known volume of N/70 sulfuric acid. The time required for distillation is from 5 to 8 minutes. The back titration is made with N/70 carbonate-free sodium hydroxide solution, using methyl red as the indicator. Satisfactory results were obtained without first boiling the acid distillate as recommended by Pregl(6), or without cooling to 15” C. as specified by Allen and Davisson (1).
PRECAUTIONS Distilled water should be freed from dissolved ammonia and carbon dioxide, or otherwise large errors would be incurred. With certain waters a satisfactory purification may only be accomplished by redistilling from an acid permanganate solution, However, it has been the authors’ experience, using distilled water from a source originally containing very small quantities of impurities, that the freeing from ammonia and carbon dioxide was accomplished most easily by vigorous
boiling for several minutes. A metal container is required for this operation, since hot water extracts appreciable quantities of alkali from glass vessels, as shown by Walther (8). Distilled water containing alkali yields high nitrogen results, as it is used to dilute the acid in the ammonia-distillate receiver. Reg1 recommended that a fused quartz tube be used in the condenser to eliminate the possibility of the steam and hot water extracting alkali from the glass. In this investigation a Pyrex tube was used and very satisfactory results were obtained, indicating that the time of contact was insufficient to incur error. Blank determinations should be made from time to time and appropriate corrections applied if necessary.
EXPERIMENTAL RESULTS To demonstrate the accuracy and practicability of the method, pure acetanilide was analyzed. The percentage of nitrogen found was 10.32, the theoretical value being 10.37. Results equally as satisfactory were obtained when using certain cereal products, as shown in Table I, the micro results being compared with those found by the Official Gunning Method ( 2 ) . TABLE I. COMPARISON OF MICRO-AND MACROMETHODS SANPLE MATERIAL MOISTURE
1 2 3 4 5 6
7 8
Flour
Flour Flour Flour Wheat
Wheat Wheat Corn
--PROTEIN ( N X 5.7)BY:Macro Micro Diff.
%
%
%
%
12.5 14.0 13.4 13.2 8.0 8.5 12.6 10.6
11.74 10.66 14.84 10.88 11.87 16.20 12.69 7.81
11.73 10.41 14.41 10.74 12.07 16.36 12.71 7.68
-0.01 -0.24 -0.43 -0.14 4-0.20 +0.16 +0.02 -0.13
The wheat and corn samples were ground on a Wiley laboratory mill (9) so that the entire sample passed through a screen of 0.5-mm. mesh. Samples 1, 2, and 5 were collaborative samples of the Association of Pacific Northwest Cereal Chemists, and consequently the macro results reported for these samples represent the averages of the protein as determined by a t least seventeen different laboratories. I n the other cases the micro results are compared with those obtained by the authors using the Official Gunning Method. On a routine basis an analysis can be made in about 35 minutes, The rapidity of this method for determining protein should be of great value to the cereal industries, inasmuch as it provides a means for rapidly checking their products. LITERATURE CITED Allen and Davisson, J. Bid. Chom., 40, 183 (1919). Assoc. Official Agr. Chem., Methods, p. 8 (1925). Bermann, Mikrochemie,2, 169 (1924). Kemmerer and Hallett, IND.ENQ. CHEY., 19, 1295 (1927). Parnas and Wagner, Biochem. Z., 125,253 (1921). Pregl, “Quantitative Organic Microanalysis,” 2nd ed., translated by Fyleman, p. 101, Blakiston, 1924. (7) Scott and Meyers, J. Am. Chem. Soc., 91,304 (1925). (8) Walther, J. prakt. Chem., 91,332 (1915). (9) Wiley, IND.ENQ.CHBM.,17,304 (1925).
(1) (2) (3) (4) (5) (6)
RECEIVBID October 19, 1931.
