presence of a fixed level of crystalline, bovine, serum albumin, and others in which a fixed amount of glucosamine was determined in the presence of varying concentrations of albumin. I n these studies the albumin was first dried in the glass-stoppered centrifuge tubes. T o the residues were added 0.5 ml. of the diluted stock solution of glucosamine and 0.5 ml. of 6 N hydrochloric acid. These mixtures mere heated and otherwise treated a s described. In the presence of 2 nig. of protein (Table 11) recoveries of about 90% were obtained for all levels of glucosamine. K h e n the protein level was raised above 5 mg., a n interference in the determination became increasingly evident. Undried and unhydrolyzed albumin had no such effect. The protein in this cnse coagulated during the heating period n4th the acetylacetone reagent. Addition of 150 y of glucose to 10 mg. of albumin, prior to hydrolysis, did not increase the interference by the protein alone. T o determine whether the drying procedure is partly related to the loss of glucosamine, 0.5 ml. of diluted stock solution and 0.5 ml. of 6-47 hydrochloric acid were dried over sodium hydroxide in vacuo. The recoveries were practically lOOyo. It is therefore apparent that the loss of glucosamine is caused by a n intrinsic interference by the pirtial hydrolyzate of the albumin. This interference does not appear to be related t o a buffering effect of the protein in the reaction with acetylacetone, as a check of the pH in reaction mixtures containing varying amounts of protein showed it to be about 9.7 in all cases. However, as the purpose of this method lies in its use with the eluates from starch electrophoresis, the expected accuracy is adequate; protein contents are generally below 2 mg. per ml. The only exception exists with eluates of the albumin peak, where the protein concentration often rises to 15 mg. per ml.
Such a high concentration of protein is present, at the most, in three eluates in a total of 20 to 30. For whole serum, the problem of protein concentration is not critical, as approximately 1 mg. is introduced with the required sample of 0.01 to 0.02 ml. With the isoamyl alcohol procedure a value of 94.5 mg. per 100 ml. was obtained for normal, human, pooled sera, which agrees with that reDorted bv Siidhof (25) and is in the Eange reported by others (5, 6 , 26). The extraction Drocedure requires Only small samples, making it potentially useful in animal studies. Electrophoretic Separation. The electrophoretic separation of 3 ml. of normal, pooled, human sera was performed on a starch block, 15 X 3 X 5 / * inches, using the general technique of Kunkel (f6). The starch block was cut into 1-cm. segments, which were then eluted n i t h 5 ml. of a 1% saline solution. Aliquots of these eluates were analyzed for their protein content by the method of Lowry (18) and for hexosamine by the isoamyl alcohol extraction procedure. The results for both protein and hesosamines are expressed as per cent of total present in the eluates. h tvpical glycoprotein
( 5 ) BOBS,N. F., J . Biol. Chem. 204, 553 (19533. Bdas, F., Bollet, A. J., Bunim, J. J., J . Clzn. Invest. 34, 782 (1955). Dische, Z., Borenfreund, E., J . Bzol. Chent. 184,517 (1950). Elson, L. A., Morgan, IT. T. J., Biochem. J . 27, 1824 (1933). Heyns, K., Koch, C. hl., Koch, \V., 2. physiol. Chem. 296, 121 (1954). Horowitz, H. N., Ikawa, hl., Fling, M , Arch. Biochem. 25, 226 (1950). Immers, J., 17asseur,E., .Vatwe 165, 898 (1950). (12) Johns,‘ R. G. s., hlarrack, J. R., Biochem. J . 5 0 , xvii (1952). (13) Johnston, J. p., Ogston, LG., i. Stanicr, J. E., Analyst 76, 88 (1951). (14) Koi\v, E., Gronnall, A , , S c u d J . Clin. & Lab. Inzest. 4, 244 (1852). (15) Bunkel, H. G., “Zone Electrophoresis,” in “Methods of Biochemical Analysis,” Vol. I, Glick, etl., Interscience, Xeu York, 1054. (16) Laurell, C . B., Skoog, X., Scand. J . Clin.& Lab. Invest. 8 , 21 (1056). (17) Leitner, J. G., Kerby, G . P., Stain Technol. 29. 257 (1954). (18) Lowry, 0. H:, Rosebrough, S . J., Farr, A. L., Randall, R. J., J . B i d . Chem. 193, 265 (1951). (19) Prodi, G., Proc. Soc. Expti. Bzol. M e d . 88, 605 (1055). 120) Rizzoli. C.. Gliozzi. AI. .4.,Boll. SOC. ;tal. bid. sner.’31. 426 i19551. (21) Rondle, C. J. hl.: Morgan, IT:T. J:, Biochem. J . 61, 586 (1955). (22) Schloss, B., ASAL. CHEM.23, 1321
clearly shows the comparatively high proportion of hexosamine in the alpha1 and alpha-2 peaks as me11 as its absence or very low concentration in albumin.
(24) Soiensen, XI., Compt. rend. trav. lab. Carlsberg 22, 487 (1938). (25) Sudhof, H.1 PetroviC, c.7 Biochem. 2. 298, 70 (1954). (26) 1yinzler, R. J , , “Determination of
LITERATURE CITED
(1) Belcher, R., Nutten, A. J., Sambrook, C. hl., Analyst 79, 201 (1954). (2) Bendich, A., Chargaff, E., J . Biol. Chem. 166, 283 (1946). ( 3 ) Bjornesjo, K. B., Scand. J . Clin. & Lab. Invest. 7, 153 (1955). (4) Blix, G., Acta. Chem. Scand. 2, 467 (1948).
s.
\
I
Serum Glycoproteins,” in “Methods of Biochemical Analvsis.” Vol. 11, Glick, ed., 1nterscie“nce: Sew York, 1955. RECEIVED for revieiv October 19, 1956. Accepted ?\larch 19, 1957. Project aided by grants from the U. S. Public Health Service [A-523(C8)] and the Michigan Chapter, Arthritis and Rheumatism Foundation
Modification of the DiacetyI Determination of Urea RALPH L. LeMAR and DAVID BOOTZIN Rock lsland Arsenal laboratory, Rock Island, Ill.
b A rapid method was needed for determination of very small quantities of urea in volatile corrosion inhibitor materials. A photometric method for estimation of urea using diacetyl as the color reagent was investigated. Modification appeared desirable and changes were made in the volume of reacting solution, reaction time, cooling time, and concentration of the color
reagent. The improved method provides increased reagent stability. The method was further investigated to determine whether certain other chemicals would interfere in the analysis.
U
is found in certain volatile corrosion inhibitor (vapor phase inhibitor) compositions. Therein, it reREA
acts with other components t o produce a volatile chemical which can sublime and be redeposited on distant surfaces and inhibit rusting thereon. A satisfactory method for urea determination applicable to small samples of these volatile corrosion inhibitor materials should require no more than 2 to 5 mg. of urea for the analysis, be rapid, and require minimum manipulation. VOL. 29, NO. 8, AUGUST 1957
1233
The classical method using urease treatment and nesslerization was found by this laboratory (4) and other workers (8-11) to be unsuitable, Orekhovich and Tustanovskii (6) developed a photometric method for urea utilizing tryptophan and biacetyl monoxime. The method, being lengthy, was unsuited for routine analysis. Watt and Chrisp ( l a ) developed a photometric method using p-dimethylaminobenzaldehyde in dilute acid as a color reagent. Exploratory work indicated difficulty in obtaining color development. Other methods (1-3) for urea determination use isonitrosopropiophenone or diacetyl monoxime in acid solution. Katelson, Scott, and Beffa ( 5 ) developed a photometric method for urea, using diacetyl in a strong acid mixture. None of these procedures was completely satisfactory for volatile corrosion inhibitor application; however, a suitable procedure was devised by minor modifications of the method of Natelson and others. EXPERIMENTAL
Initially, the maximum absorbance was confirmed to occur a t 480 mp, when a Beckman Riodel DU spectrophotometer was used. Then the volume of reacting material was increased to reduce volumetric errors. The concentration of diacetyl in acid mas reduced to provide greater reagent stability, color stability, and optimum color intensity rate of change per change in urea concentration. As the reaction producing the yellow complex is accelerated by heating, this factor was studied. Maximum color stability occurred between 19 and 21 minutes of heating time for four different urea concentrations. A cooling period of 10 minutes proved adequate. Improved Method. Acid mixture, a 1:3: 4 mixture of concentrated sulfuric acid, 8570 phosphoric acid, and water. Diacetyl in ethyl alcohol (5% by volume). Acid diacetyl reagent, 1.0% by volume of diacetyl in ethyl alcohol solution in the acid mixture. This solution is stable for several weeks if protected from light. Urea Stock Solutions. Stock standard A, 1.00 gram of reagent grade urea in 1000 ml. of water. Stock standard B. Dilute 50 ml. of stock standard A to 500 ml. with water. Solutions A and B are stable for approximately 2 to 3 weeks when stored a t 4" to 6" C. and preserved by 1 ml. of chloroform.
1234
b
ANALYTICAL CHEMISTRY
Working standard solutions containing 15, 20, 25, 30, 35, 40, and 45 y of urea per milliliter are prepared from stock solution B. A few drops of chloroform are added to each solution. These solutions must be used the same day as prepared.
extracts containing sodium nitrite, which interfered in the accuracy of results. The following relationship corrects for such interference when the nitrite concentration is known:
cu
PROCEDURE
Five milliliters of each working standard solution are pipetted into separate test tubes (2 X 14.5 em.). Five milliliters of the acid-diacetyl reagent are added to each and the mixtures are agitated. Air condensers (3//16 X 4 inch glass tubes in corks) are placed in the test tubes. Within 5 minutes of diacetyl reagent addition, the test tubes are immersed in a boiling water bath for two thirds of their length. The mixture is protected from light during heating. The water should resume boiling within 1 minute of test tube insertion. A tripod arrangement supporting a perforated metal plate serves as a test tube holder. This assembly fits into a stainless steel beaker which protects the tests from light. The mixture is heated for 20 f 1 minutes. Immediately thereafter, the tests are placed in a second water bath (initially a t 4" to 6' C.) for 10 minutes. There should be a t least 100 ml. of coolant for each test tube. The mixtures are agitated and per cent transmittance values a t 480 mp are determined. The per cent transmittance values are plotted against urea concentration values (7per ml.) on semilogarithmic graph paper. With this relationship, urea concentration can be determined in unknowns. Five milliliters of any solution containing from 15 to 45 y of urea per ml. are treated as indicated above. The per cent transmittance values of the unknown solution are converted to urea concentration values by use of the prepared curve. METHOD EVALUATION
The method was examined for repeatability of results. Urea solutions were prepared and each of two operators carried out three series of tests, using the same color reagent. Three measurements were made on each solution. The results indicated that the reproducibility of the method had an average coefficient of variation of 0.917%. This analysis was also applied to
=
Cua
1 - 0.0046 ( C N ~ N O ~ )(1)
where
Cu
= true concentration of urea Cua = apparent concentration of urea after sodium nitrite interference
A volatile corrosion inhibitor paper containing only urea and sodium nitrite was analyzed for its inhibitor content; chemical methods were compared with a determination of total water-soluble solids. The sodium nitrite was determined by a colorimetric procedure, originally described by Shinn (7) and modified for volatile corrosion inhibitor papers by Johnson and LeMar (4). The urea was determined by the procedure described above. The chemical methods gave results differing from the water extraction method by 5Y0 or less besides providing the needed detailed information about the composition of certain volatile corrosion inhibitor papers. LITERATURE CITED
(1) Archibald, R. M., J . Biol. Chem. 156, 121-42 (1944). (2) Barker, S. B., Ibid., 152, 453-63 (1944). (3) Fearon, W. R., Biochem. J . 33,902-7 (1939). (4) Johnson, R. E., LeMar, R. L., Rock Island Arsenal Lab. Rept. 53-2670 (1953). (5) Natelson, S., Scott, M. L., Beffa, C., Am. J . Clin. Pathol. 21, 275-81 (1951). (6) Orekhovich, V, , N., Tustanovskii, A. A., Bzokhzmzya 14,444-8 (1949).
(7) Shinn, Martha, IND.ENG. CHEM., ANAL.ED. 13, 33 (1941). (8) Sumner, J. B., Proc. SOC.Ezptl. Biol. Med. 24, 287-8 (1927). (9) Sumner, J. B., Hand, B. B., Holloway, R. G., J . Biol. Chem. 91,333-
41 (1931). (10) Sumner, J. B. Myrback, Karl, 2. physiol. Chem., 1.89, 218-28 (1930). (11) Sumner, J. B., Sisler, E. B., Arch. Biochem. 4, 207-10 (1944). (12) Watt, G. W., Chrisp, J. D., ANAL. CHEM.26, 452-453 (1954).
RECEIVED for review Kovember 3, 1956. Accepted March 14, 1957. The opinions or assertions contained herein are not to be construed as being official or reflecting the views of the Department of the Army.
