Colorimetric Determination of Nitrogen in Biological Materials JOHN R. POLLEY Virus Section, Laboratory of Hygiene, Department of National Health and Welfare, Ottawa, Can.
T
HE Kjeldahl method for the determination of nitrogen has
been undergoing constant investigation and revision to increase the accuracy of protein nitrogen determinations and to extend the method to other forms of nitrogen. The present investigation was undertaken to develop a method of nitrogen determination which would be applicable to problems on tissue protein fractionation involving numerous determinations per day. Most methods require some prior knowledge of approximate nitrogen content and the size of sample used is selected so that the final ammonia value will be within a given range of titration or color intensity. I n the proposed problem it was expected that the range of nitrogen content in various samples would be large and unknown for each sample. The digestion mixture selected for maximal conversion of nitrogen to ammonium sulfate was similar to that of Miller and Houghton ( 4 ) except that the mercury was added in the form of mercuric sulfate solution. This digestion mixture, however, does not lend itself directly to nesslerization because the mercury present causes precipitation with the Sessler reagent. Recently, Hiller et al. ( 2 ) in their titrimetric procedures used zinc to amalgamate with the mercury. Hence, i t was proposed to digest the samples with the selected digestion mixture, then to add zinc dust to remove free mercury, thereby making possible a colorimetric assay of the nitrogen content. REAGENTS
Reagent grade chemicals were used. Mercuric Sulfate, 10%. 12 ml. of concentrated sulfuric acid are diluted to 100 ml. with distilled water and 10 grams of mercuric oxide dissolved in it. Nessler Reagent. This was prepared as directed from the commercially available dry salt, Nessler Compound-Paragon (Paragon C. & C. Co., Inc., New York 58). Nitrogen Standards. Ammonium sulfate (14.7469 grams), dried for 1 hour a t 110" C., is dissolved in distilled water and the volume made up to 250 ml. This solution contains 1250 mg.% nitrogen and other standards are prepared from this by dilution. Potassium sulfate, powder. Sodium hydroxide, 5N. Sodium hydroxide, 1N. Sulfuric acid, concentrated. Zinc, dust.
after 24 hours, the color intensity had increased only slightly, that a reading made a t this time indicated a nitrogen content which was 4% higher than the original recording. The nitrogen content is estimated from a standard curve between colorimeter readings and nitrogen content, obtained by treating a series of nitrogen standards, 0 to 1000 mg.% nitrogen, in the same way throughout. This is referred to as the general curve. Macro Determination of Nitrogen. If the nitrogen content of a sample is found to be greater than 750 mg.%, , precision is increased by making a 1 to 1 dilution of the digestion solution and repeating the color development on a second 0.5-ml. aliquot. The nitrogen content is determined from a standard curve, referred to as the macro curve. Micro Determination of Nitrogen. If the nitrogen content is found to be less than 100 mg.70, the following procedure is used to obtain a more accurate determination. T o a 1.5-ml. aliquot of the same digestion solution is added 1.5 ml. of 1 N sodium hydroxide and the tube is placed in ice water for approximately 5 minutes. The 1.5 ml. of Nessler reagent is added and the color intensity determined after 15 minutes. If any gelatinous precipitate is present, the tubes are centrifuged and the supernatant liquid is used for the intensity measurements without impairing the results. The nitrogen content of the sample is then determined from a standard curve, referred to as the micro curve. SO
Precision of the Method. T o determine the precision of the method, replicate samples (6 to 10 of each) of a series of nitrogen standards were analyzed by these procedures and the respective nitrogen values obtained from the standard curves. The results are shown in Table I. Determination of Nitrogen in Amino Acids. I t has been reported that it is relatively difficult to obtain quantitative recovery of nitrogen from certain amino acids, notably lysine ( 4 ) ,histidine, and tryptophan ( 3 ) . Consequently, aqueous solutions of known nitrogen content were prepared from various amino acids which were first dried a t 110" C. for 1hour. These solutions were then analyzed and the results are shown in Table 11. From Table I1 it can be seen that within the =t2% experimental error of the method, there was complete recovery of the nitrogen from the various amino acids used with the exceptions of
Table I. Xitrogen Content of Standard, llg
PROCEDURES
Digestion. Into a test tube of borosilicate glass, 25 X 200 mm. (Folin tube), calibrated at 15 ml., is placed 1 ml. of distilled water followed by 0.2 ml. of sample and the pipet is rinsed with the contents of the tube. Then 0.5 gram of potassium sulfate, 0.5 ml. of mercuric sulfate solution, and 1.5 ml. of concentrated sulfuric acid are added. A glass bead is added and boiling is begun and continued gently for 30 minutes. S o special care is taken to prevent white fumes from escaping from the tube. The tubes are allowed to cool slightly and the sides of the tube are washed down with 5 ml. of distilled water. T o amalgamate the mercury, 0.2 ml. of zinc dust is added to each tube and the mixture is heated gently until the visible reaction of the zinc with the solution has ceased (2 to 3 minutes). The mixture is allowed to cool slightly and 7.5 ml. of 5;V sodium hydroxide are added with continuous shaking. The tubes are cooled to room temperature and the volume made up to the 15-mI. mark with distilled water. This solution is referred to henceforth as the digestion solution. Preliminary Nitrogen Determination. To a colorimeter tube or test tube containing 10 ml. of distilled water is added 0.5 ml. of the digestion solution. The tube is placed in ice water for approximately 5 minutes, then 1.5 ml. of Nessler reagent are added. The tube is stoppered and left in the ice water. The color intensity is determined in a photoelectric colorimeter a t 450 mp after 15 minutes. (The instrument used was a Coleman Junior spectrophotometer, Model 6B.) Under these conditions, the color was quite stable for samples up to 500 mg.% nitrogen, Even
%K
10 50 100 100 250 500 750 750 1000 1250
Analytical Precision Obtained by Replicate Analyses of Nitrogen Standards Standard Curve Used Micro Micro hlicro General General General General Macro Macro Macro
Range of Values Found 8.0-10.8 48-52 98-102 99-101 245-255 490-507 730-750 740-770 990-1028 1220-1260
Average (i.S.D.) 9.6 i 1.0 49.7 i 1.8 99.2 i 2 . 4 99.6 i2 . 2 252 i 4 . 6 500 i 5 745 = 8 754 zt 12 1003 zk 16 1244 zt 17
Table 11. Recovery of Nitrogen from Various Amino Acids Amino Acid Z-Phenvlalanine Z-PheniManine 1-Arginine hydrochloride I-Arginine hydrochloride DL-.ilanine Z-Cysteine hydrochloride Z-Cysteine hydrochloride [-Glutamic I-Glutamic 1-Lysine l-Lysine I-TrrDtonhan I-TrGbtophan I-Hiitidine hydrochloride [-Histidine hydrochloride
1523
Sitrogen Content of Sample, Mg. % ' 200 50 200 50 200 50 200 50 200 50 250 50 200 50 250 50
Sitrogen Found (iS.D.),
vi&%
202 i 2 48.9 i 1 . 5 198 i 2 49.8 f 1 . 2 201 i 1 51.2 i 1 . 6 198 i 2 49.4 i 1.0 199 i 2 49.2 i 1 . 2 252 i 2 50.5 i 1 . 4 200 3 -48.5 Z i . 7 244 zt 2 47.0 1.6
*
+
ANALYTICAL CHEMISTRY
1524
tryptophan and histidine, both of which are heterocyclic. With the heterocyclic compounds the recovery of nitrogen was 94% or greater. F~~~the amino acid composition of a number of proteins ( I ) , it appears that in general about 10% or less of the r,itrogen presentis in heterocyclic compounds. on the basis of the author's experiments it would be expected that the total nitrogen unrecovered by this method would be in the order of 1%. H ~ protein ~ standard ~ ~ solution , (a4rmour & co,,Chicago, 111,, 9.8 mg. protein K/cc.) was analyzed. Determinations were made on eight samples of this solution. The range of values found was 965 to 1000 mg. % nitrogen with an average Of 980 mg. %, standard deviation &16, standard error zt6.
LITERATURE CITED
(1) H a u r o w i t r , Felix, "Chemistry a n d Biology of P r o t e i n s , " p. 3 2 , New Y o r k , A c a d e m i c Press I n c . , 1950. (2) Hiller, A%., PlaZin, J., a n d V a n S l y k e , D. D., J . B i d . Chein., 176, 1401 (1948). (3) Jonnard, R., IND.ENG.CHEM.,A N A L .ED., 1 7 , 246 (1945). (4) A1iller3 L., a n d H o u g h t o n , J. A . , J . Bid Chem., 159, 373 (1946). RECEIVED for review March 12, 1954. Accepted June 3, 1954. Presented before the Division of Bnalytical Chemistry a t the 124th Meeting of the h f E R I C A S CHEMICAL SOCIETY, Chicago, I11 , September 1953.
CRYSTALLOGRAPHIC D A T A
86. Tris(ethylenediamine)cobalt( Ill) Phosphates Contributed by HOMER W. MCCUNE and Proctor 81Gamble Co., Cincinnati 31, Ohio
NED WILKINS, M i a m i Valley Laboratories,
CO(NHZ-CZHI-?;HZ)~HZP~O~O. 2Ha0 TRIPHOSPHATE DIHYDRATE C o ( S H p C a H r - ~ H z ) a H P ~ O H:O :. Co(iVHpCtH~--lu"~) JHP?OI PYROPHOSPHATE PYROPHOSPHATE MONOHYDRATE ANHYDROUS
6.5. The crystallographic data given can be used to identify the precipitates. Table 11. X-Ray Diffraction Powder Data, Cr Kcu Radiation Co(enjsHzPaOu.2HzO d I/Ii 1 12 2 9,4 1 8 6 7 7 8 2 6.51 5.92 10 5.32 9 4.71 9 2 3.94 3 80 ? 3.67 3 . .54 1 3.40 4 3.28 2 3.15 1 3.0(i 4 2.96 3 2.75 2 2.63 2 2.59 2 2.40 1.95 2
Abbreviated Structural Formulas HE crystallographic data reported in Tables I and I1 were determined as part of a larger program to find specific reagents for the determination of condensed phosphates. The reagent tris(ethylaminediamine)cobalt( 111) chloride is a specific precipitant for triphosphate a t pH 3.5 and precipitates pyrophosphate a t pH
Table I. Crystal system Optical properties Refractive indices (5893h; 25' C . )
OpticYaxial angle,
Co(en)aHP?O:. HzO Triclinic
1 , 5 8 0 f 0.001 1.583 i 0.001 1.584 i 0 . 0 0 1 Ca.82' (univ. stage)
u
B
zv
Sign of double refraction Dispersion Extinction angle
Negative See extinction angle pa tablet face = 36O (6563 .I.), 38' (5893 .I ) , 42O (4861 A , )
1.609 + 0.001 1 . 6 2 8 z 0.001 1.637 z 0 . 0 0 1 79' (Berek's method) Kegatire Very slight y'A edge = 2' on tablets ,showing y ' and a
\
\
\ \
9
I
3 1 4 1
/
/ /
/
/
'\ \
\.
I
--P+
&'
TI
--b
1 1
I
I
I I
ij
m
--?
I -L I I
\
2.8ii
2.79 2.7: 2.60 2.31 2.24 2.18
Co(en)aHPzOr d I/Ii 10 8.3 7.6 6.7 6.04 5.41 4,81 4.36 4.00 3.74 3.46 3.12 2.94 2.84 2.72 2.59 2.48
CONTRIBUTIOSS of crystallographic data for this section should be sent to Walter C. RIcCrone. Analytical Section, .Irmour Research Foondation,Illinois Institute of Technology, Chicago 16, Ill.
I 38'
; :
:
Optical Properties Co(enjsHzPaOio.2HzO IIonoclinic
Co(en)sHP~Or. H S d I/Il 2 9.3 10 8.4 !I 7.2 6.59 ! 6 . 0fi 5.78 3 3 4.99 4.75 4.34 4.01 3.70 4 3.60 3.50 5 3 3.40
r'= a
I I I I
l'
II
- I1
I
I I
j
< T q - t - - b
____
Exo I
B
A
Figure 2. Figure 1. Orthographic Projection of Typical Crystal of Co(en), H?P30m. 2IIzO
Co(en)sHPzO,.H20
a. Perspective v i e w
b.
orthographic projection of c o m m o n view
