drnong phosphorus content of the grain and the various quality measures was toward improved malting quality. All correlations of phosphorus content of the grain with the other criteria were positive. and although most of the coefficients were not sufficiently great to indicate a pronounced increase in the qualit\ criteria from applying phosphorus fertilizer. the) were in the desired direction. The associations of protein content of the grain and malt with other quality factors Ivere more variable than the similar associations betrveen phosphorus content and the same factors. Both negative and positive correlations were observed for the relationships with protein content. but again the coefficients did not appear to be sufficiently high for most comparisons to constitute a serious deterrent to good quality. The negative correlations bet\\een protein content and malt extract percent and bet\\een protein content and 1000-kernel weight were the most undesirable from a qualit1 standpoint. Acknowledgment The authors are indebted to L. B. Nelson. formerly associate professor of soils. Iowa .4gricultural Experiment
Station, who initiated the study, and to R . P. Nicholson, research associate in soils, for assistance ivith the fieid tvork in 1949 and 1950. Further gratitude is acknowledged to .2. J. Lejeune and J. H. Parker, Malting Barley Improvement Association: A. D. Dickson, Barley and XIalt Laboratory, Madison? \Vis., L. .4. Hunt, Joseph Schlitz Brewing Co., Milwaukee, Wis., and T. Ehret. Froedtert Grain and Malting Co., Milwaukee, \Vis., for helpful suggestions in evaluating the effects on malting quality. Literature Cited (1) Ahr, J.. and Mayr. C., E r p . S t a . Record? 46, 729 (1922). (2) Anderson. J. L4.. Sallans. H. R.: and Meredith, W. 0. S..Can. J . Research, 19C, 278 (1941). (3) Burkhart. B. A,: and Dickson. A . D.. Barley and Malt Laboratory Report, Madison, Wis.. 1946. (4) Den Hartog. G. T., and Lambert, J. W., Agron. J., 45, 208 (1953). (5) Foote, W. H., and Batchelder. F. C.? Zbid.,45, 532 (1953). (6) Frey, K. J.: and Robertson, L. S., Cereal C h e m . , 30,31 (1953). ( 7 ) Frey? K. J.: Robertson, L. S., Cook, R. L.: and Down, E. E., Agron. J.: 44, 179 (1952).
Gregory, F. G.. and Crowcher E. A,. =inn. Botany 42, 7 j 7 (1928). Hanway. J.. and Dumenil, L. C.. Soil Sci. Sac. .-lmer. Proc.. 19, 77 (1955). Merediih. \V. 0. S.:Sci. A g r . . 23, 355 (1943). hleredith, W. 0. S.:and An lerson. J. A.: C a n . J . Research, 16C, 497 (1 938). Meredith, \V. 0. S.. Olson, P. J., and Rowland. H.: Sci. Agr., 23, 135 (1942). Meredith, W. 0. S., Sallans, H. R.! and Rowland, H., Ibid.,22, 761 ( 1942). Neatby, K. W.!and McCalla. A. G., Can. J . Research, 16, 1 ( 1938). Olson, P. J., Meredith, W.0. S.: Laidlaw, H. C.: and Lejeune, &4.J.: Sci. A g r . ? 22, 659 (1942). Pendleton, J. W., Lang, A. L., and Dungan, G. H., Agron. J., 45, 529 (1953). Stanford, George, and Hanway, J., Soil Sci. Sot. A m e r . Proc., 19, 74 (1955). Receioed f o r review February 17, 1955. Accepted M a y 9, 7955. Study supported i n part hy a grant-in-aid f r o m the ‘Malting Barley Improvement Association, M i l u aukee, W i s . Journal Paper J-2702, Project 1149, Iowa .igricultural Experiment Station, Ames, Iozea.
AGRICULTURAL PRODUCTS ANALYSIS
G. C. ELLIS and R. 1. FORMAlNl Nitrogen Division, Allied Chemical and Dye Corp., Hopewell, Va.
Colorimetric Determination of Biuret
Previous methods for biuret determination-i.e., complexing with copper ion in alkaline media-necessitate removal of hydrated oxide precipitates prior to measurement of the color development. These methods were found to be unreliable and the inaccuracies were attributed to such factors as sorption of the colored complex b y the oxides formed and variance in reagent concentration. The procedures were modified and specifically developed for urea pyrolyzate products containing biuret in a wide concentration range. This new method is based on an advantageous equilibrium existing between coppertartrate and copper-biuret complexes in Fehling’s solution, The equilibrium favors the more highly colored copper-biuret complex, oxide precipitation i s prevented, and biuret can be directly determined b y standard colorimetric methods.
B
in agriculture as a combined herbicidefertilizer and in the resin industry as a n intermediate. It may be conveniently prepared by pyrolysis of urea a t moderate temperatures. The solid pyrolyzates contain varying proportions of concomitant materials such as urea! cyanuric acid, ammonia, and triuret. The development of direct analytical methods for biuret was based on proIURET FINDS POTENTIAL USE
cedureszapplicable to materials containing the polypeptide linkage or giving the “biuret test” (2-4, 6). These methods entail addition of excess sodium hydroxide to dilute copper sulfate solutions of biuret? which results in the development of a colored copper-biuret complex and precipitation of hydrated copper oxides. Oxide removal. by filtration, provides a solution of the complex suitable for colorimetric analysis by
conventional methods. The accuracy and precision of these methods were found to be limited by such factors as the sorption properties of hydrated oxides, peptization phenomena, and effects promoted by variance in reagent concentration (Table I). Experimentation revealed that reversing the order of reagent addition-i.e., addition of copper sulfate solution to biuret solutions of fixed sodium hydroxide
VOL. 3, NO. 7, J U L Y 1 9 5 5
615
concen tration-results in a n immediate development of color, with oxide formation apparent only after near depletion of uncomplexed biuret. Addition of a n excess of reagent terminates in formationof peptized oxides and suspensions unsuited for colorimetric analysis (Figure 1). Incorporating Rochelle I salt in the system , prevents oxide for75 El-30 0 0 . B I U R E T PkR 100 .I, mation, yielding A-SO mo. B I U R E T PER 100 .I, I 0-100 MI. B I U R E T E R 100 m l solutions of colored complexes ideal for 0 0 0.1 0.2 0.3 0.11 0.1 0.6 0.7 direct colorimetric CONCENTRATIOU O F NaOH - MOLES PER L I T E R determination (7). Over the biuret conFigure 1 . Effect of sodium hydroxide concentration on centration range emcolorimeter reading ployed. 10 to s"0mg. per 100 ml.. the system conforms with T h e coordinating characteristics of cupric Beer's 1aj.r.. Slight deviations are enion allow prediction of possible intercountered a t loiver and higher biuret complexing in the system. concentrations (Figure 3), but d o not appear to reduce the precision or accuracy Reagents and Apparatus of the method. T h e procedure was 1. Copper Sulfate Reagent. Dissolve adapted to the Klett-Summerson photo15.0 grams of reagent grade cupric sulfate electric colorimeter. glass cell model, using pentahydrate in distilled water, dilute to a 40-mm. cell path and a S o . 54 green 1.0 liter, and age for 1 day before using. filter. This solution is 0.06M in copper sulfate. T h e reagent and biuret concentrations 2. Alkaline Rochelle Salt Reagent. employed were satisfactory for the Dissolve 50.8 grams of reagent grade sodium authors' purposes, but do not necespotassium tartrate tetrahydrate in distilled water containing 51.4 ml. of 50% sarily represent the optimum range sodium hydroxide (carbonate-free), dilute applicable to the method or other instruto 1.0 liter, and age for 1 day before using. ments. I t is beyond the scope of this Solution is 1.ON in sodium hydroxide and paper to postulate whether the system 0.18.2.1 in tartrate salt. represents a true copper-biuret and 3. Distilled water, 0.llVsodium hydroxcopper-tartrate equilibrium, o r the ide and sulfuric acid, prepared by known formation of a biuret-copper-tartrate methods. complex of undetermined structure. 4. Biuret Standard Solution. A suit-
'7
Table 1. Grams Solid per
Run
IO00 MI.
No.
Solufion
Urea
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
20.023 9,870 8.961 4.754 4.960 4.894 5.142 2,518 5.055 2.459 3,952 2.553 2.445 2.489 2.050
80.0 73.9 71 . O 60.8 51.9 44.8 42.1 34.9 31.6 25.7 21.1 7.7 5.5 3.1 1.3
able anhydrous reagent material may be prepared by crystallizing Eastman white label biuret successively from water. 1 N sodium carbonate, water, and 95% ethyl alcohol ( 5 ) . The standard solution is prepared by dissolving a weighed sample of the reagent biuret ( h 0 . 2 mg.) in distilled water, neutralizing to a pH of 7.0, and diluting to 1.0 liter. For convenience the solution should contain 2.0 mg. per ml. 5. Water Bath. A thermostated bath set to operate at a fixed temperature (zkl.0' C.): preferably in the25' to 35" C. range. 6. Colorimeter. A Klett-Summerson photoelectric colorimeter, glass cell model, 40-mm. cells, and No. 54 green filter (490 to 570-mp spectral range). 7. pH Meter, glass electrode type. Analytical Procedure
Preparation of Samples for Analysis
Samples Soluble in Water at 25" C. Dis-
solve a Iveighed, pulverized sample ( 5 0 . 2 mg.) containing 0.4 to 1 8 grams of biuret in 500 ml. of water. Neutralize the solution with 0.l.l' acid or base to a p H of 7.0, transfer to a 1000-ml. volumetric flask, and dilute to the mark. Transfer a 50-ml. aliquot of the solution to a 100-ml. volumetric flask and subsequently treat bv the procedure for "complex formation." Samples Insoluble in Water at 25 " C. Transfer a weighed. pulverized sample (=k0.5 mg.) containing 0.4 to 1.2 grams of biuret to a 1000-ml. beaker with about 700 ml. ofwater. Heat the suspension to 70" to 80" C. with stirring, and air-cool to 30" C Neutralize the solution to a pH of 7.0 using 0.l.l' acid or base and filter into a 1000-ml. volumetric flask. Il'ash the residue Ivith three 50-ml portions of water. combine washings and filtrate. and dilute the solution to the mark.
Colorimetric Determination of Biuret in Urea Pyrolyzate Mixtures Solids Cornposifion, Wf. % Cyanuric Biuref acid
14.3 15.2 15.1 14.8 22.8 29.7 34.8 14.8 24.4 5.5 10.0 10.7 10.1 10.4 5.5
Triuref
Calcd.
4.0 7.8 8.9 17.5 14.9 10.5 3.4 25.2 8.3 28.7 18.6 21.6 15.0 8.8 1.8
17.0 l5,3 22.4 16.4 25.8 36.7 50.6 31.6 90.2 49.3 99.4 76.6 88.2 96.7 93.7
Observed Mefhod 1 Mefhod 2
70 Biuref
Recovery
Mefhad I
16.8 16.9 98.9 15.0 15.3 98.0 21.6 22.2 96.4 16.5 97.5 16.0 24.0 25.9 93.0 36.8 36,6 100.3 50.2 50.1 99.2 32.0 97.6 32.9 89.1 98.8 90.3 47,l 49.9 95.6 97.5 100.0 98.1 76.6 77.1 100.0 83.6 88.2 94.8 95.0 96.5 98.2 90.6 93.5 96.7 Arithmetic mean 97.5 Average deviation 1.5 Method 1. Add 2.0 ml. of 20% NaOH to solution containing 25 ml. of O.06M C u S 0 4 and 50 ml. of test solution, dilute volume, filter free of hydrated oxides. Color by Klett-Summerson colorimeter: 20-mm. cell path, green filter (No. 54). Method 2. Standard procedure.
616
1.7 3.1 5 .O 6.9 10.4 15.0 19.7 25.1 35.7 40.1 50.3 60.0 68.4 77.7 91.4
M q . B i u r d per 5 0 - M I . Aliauaf
AGRICULTURAL A N D FOOD CHEMISTRY
Mefhod 2
99.4 100.0 99.1 100.6 100 4 99.7 99.0 101.3 100.1 101.2 100.6 100.7 100.0 99,8
99.8 100.1 0.5 to 100 ml.
Transfer a 50-ml. portion of the solution 10 a 100 ml. volumetric flask and subsequently treat by the procedure for comd e x formation.
Complex
Standard
Procedure.
Successively add from Formation burets 20.0-ml. portions of alkaline tartrate and copper sulfate reagents with constant sxvirling. Dilute the colored solution to the mark. shake vigorously, and suspend in the therniostated bath for 15 to 30 minutes. Shake occasionally during this aging process. Then remove the solution from the bath and determine its colorimetric scale reading mith the photoelectric colorimeter. Set the water bath Preparation of to operate a t a fixed Standard Curve temperature ( 51.O 0 C..) in the 25 to 35 C. range. Transfer from a 50-ml. buret varying portions of biuret standard solution (0 to 50.0 ml.) to 100-ml. volumetric flasks. Adjust total ~ o l u m e sto 50 ml. Ivith water and subsequently treat as in the procedure for complex formation. Take colorimetric readings, using either a reagent or t\ater blank. .4 rectangular plot of colorimeter stale reading LS. milligrams of biuret per 100 ml. of solution gives the standard curve, Ivhich is linear in the range of 10 to 80 mg. of biuret per 100 ml. of so1utio:i. For any set of colorimetric reagents the curve is fixed and may be used for extended periods. In this laboratory, a plotting scale of 5 mg. of biuret and 25 colorimeter scale units per inch was found most satisfactory. Linear plotting is permissible. as the Klett-Summerson colorimeter scale is logarithmic and directly pro;lortional to absorbance.
Calculaiions Using the colorimeter scale reading, the biuret concentration in the aliquot of sample solution is obtained directly from the standard curve. Then, ( M y of biuret)
filtrations necessary in other metliods, reduces operational time, and increascs the precision and accuracy obtained in colorimetric drtermination of biuret. For compai ison. biuret was determined in kno\\ n urea pyrolyzate compositions,
(volume of sample solution, ml.) (100 1
~
,,
[,
(1000) (sample weight, g.) (aliquot, ml.)
Discussion
In developing this method of analysis. consideration \vas given to water solubility of urea pyrolyzate, biuret concentration, and acceptable colorimeter scale limits. Based on the solubility of the least soluble component, a concentration of 2.0 grams of urea pyrolyzate sample per liter \vas selected as the upper limit. This fixed the biuret content, per 50-ml. aliquot, in the 0- to 100-mg. range. As maximum absorption of transmitted light by copper-tartrate-biuret solution occurs at ca. 550 my. a green filter (490 to 570 mp) \vas required with the KlettSummerson colorimeter (Figure 2). .4ccurate scale readings on this colorimeter can be made in the 0 to 400 unit range (
