November. 1923
INDUSTRIAL A N D ENGINEERING CHEMISTRY
1179
Nitrogenous Studies of the Activity of Organic Compounds-I I’j2 By C. S. Robinson, 0. B. Winter, and Selma Bandemer MICHIGAN AGRICULTURAL EXPERIMENT STATION, EASTLANSING, MICH.
HIS work may per-
tube containing acid was I n a preuious publication3 i f was shown that a more or less intimate haps be characterized then made and 40 cc. of an relationship existed between the nitrogen present in an acid hydrolyalkaline permanganate soluas an analysis of the sate as amino acids and acid amides and that evolved as ammonia tion (one-third the official a 1k a 1i n e permanganate upon oxidation with alkaline permangunate solution as prescribed quantity) were introduced method. In it an attempt by the official methods of the A . 0. A . C.4 has been made t o follow through the inlet tube, I n the present article the action of permanganate is further the progress of the digestion slight suction being applied studied using two samples of protein and several of common base and distillation by analyzto the outlet of the acid goods of the fertilizer trade. Previous work dealt only with the ing the digestion mixture a t tube for the purpose. The ultimate quantities of ammonia, acid amide, and amino nitrogen regular intervals and by reaction tubes were then obtained upon decomposition. I n the work herein reported an collecting the distillate in thoroughly shaken and set effort was made to ascertain whether or not any connection exists fractions in each of which in a water bath (95OC.) between the rates of decomposition of various materials and their while the attached tubes the ammonia was deterfertilizer oalues. The study of the several factors influencing the mined. were held outside of the results with the permanganate digestion was also undertaken. The procedure finally folbath bv means of clamm. I n working with pklowed was the outcrrowthof a lengthy study of ;he problem and of many attempts to de- teins it was found that complete solution was effected velop a method as closely analogous as possible to the official during the 15 minutes allowed for the temperature of the technic. One plan tried out involved running the experi- contents of the tubes to rise from that of the room to ments in regular Kjeldahl flasks and sampling the contents that of the bath. This procedure was arbitrarily followed at incervals. It was thought that satisfactory results could with all the other materials, assuming that the alkalibe obtained by assuming that the total nitrogen present soluble matter would be dissolved during this prelimiin the system at any time equaled the amount originally nary stage. Time was counted from this point and one present less the ammonia removed. Only after consider- tube removed every 5 minutes for 30 minutes, an interval able work had been done and a number of other possibilities corresponding to that specified for the digestion of the eliminated was it discovered that nitrogen escaped in some sample in the official A. 0. A. C. methods. Each tube was form other than ammonia (apparently as free nitrogen), removed with the acid-containing one attached and imwhich fact introduced appreciable errors into the results. mediately cooled to room temperature to stop the reaction. It W A S found necessary actually to determine in each frac- The outlet of the acid-containing test tube was then attion both the ammonia in the distillate and the total nitro- tached to a suction pump to remove any ammonia remaining gen in the residue. in the digestion mixture and the excess acid titrated as usual. The digestion residue was transferred to a volumetric APPARATU s flash, acidified with sulfuric acid, diluted to the mark, and The apparatus consisted of twelve pairs of test tubes, used for the determination of total and amino nitrogen. At the end of the 30 minutes, when the sixth pair of tubes 2.5 cm. in diameter and 25 cm. long, each tube being fitted with a perforated rubber stopper through which passed two had been removed, the remaining six were transferred to pieces of glass tubing about 6 min. in diameter. One of an oil bath. After 10 minutes one tube was taken out and these reached almost to the bottom of the test tube and another a t the end of each subsequent 10-minute interval, terminated in a bulb perforated with a number of holes which so that the total time consumed in the experiment, exclusive servod to break up into small bubbles the air stream drawn of the preliminary “solution” period, was 90 minutes. The through it, while the other was flush with the bottom of the oil bath used during the last 60-minute stage was held a t stopper. One test tube in each pair served as a reaction such a temperature (140’ C.) that only a small amount of vessel and the other as a receptacle for standard acid to liquid remained in the last tube when it was removed. By catch the ammonia evolved, the outlet tube of the former this procedure its contents were subjected to the conditions prescribed by the official methods. connecting with the inlet tube of the latter. It must, of course, be admitted that this procedure, which PROCEDURE differs markedly from that specifically fixed in the official In making the determinations one-third of the amount of method, may in some cases lead to very different results. sample necessary to furnish 50 mg. of water-insoluble ni- The rather good agreement between the final values for the trogen-i. e., one-third of an official sample-was weighed active insoluble nitrogen as determined by the two procedures into each reaction tube. The connection with the companion indicates that such differences are but little in excess of those normally to be expected with the official method. The 1 Received July 27, 1923. writers, therefore, feel confident that, in spite of the radical 2 Journal Article No 25 from the Chemical Laboratory of the Michigan differences in technic, the conclusions drawn from the present Agricultural Experiment Station. Published b y permission of the Director work are applicable to the official procedure. of the Experiment Station. The material contained in this article was originally to be published in two papers, the secopd and third in the series. As All determinations were made in duplicate and the duplisuch they were read by title at the Birmingham and Pittsburgh meetings, cates agreed as well as could be expected. respectively, of the American Chemical Society. For purposes of comparison, determinations using the same THISJOURNAL, 13, 933 (1921). strength of alkali but omitting the permanganate were also $1 Assoc. OfficialAgr. Chem.. Methods, 1920, p. 11.
T
$1
Vol. 15, No. 11
Ih-DUSTRIAL A N D ENGINEERTNG CHEMISTRY
1180
90 80
70 60
50 40
30 25
PO 15
IO
f
3-
(full Sample) 5 FIG. 3
FIG. 1
FIG.2
made, thus making it possible to calculate the actual results due to the permanganate. The results are shown in the accompanying figures.
EFFECTOF ALKALIALONEAND OF ALKALINE PERMANGAKATE The chief result of the action of alkali on protein compounds is one of hydrolysis with the formation of ammonia and amino acids, the former being produced chiefly from acid amides, although there are some secondary reactions whereby some compounds other than acid amides are made to yield ammonia. While the relative amounts of ammonia and amino nitrogen produced by protein hydrolysis vary with different substances, the quantity of amino nitrogen is always much greater than the ammonia. In the most common animal proteins the ammonia formed during acid digestion amounts to about 5 to 10 per cent of the total nitrogen, while the amino fraction approximates 60 to 70 per cent. Data are not available on the alkaline hydrolysis of many materials, but these figures would be somewhat changed, the ammonia tending to increase a t the expense of the amino nitrogen. Inspection of the graphs illustrating the results of the action of alkali alone upon the materials used in the present work shows that the quantities of ammonia are unusually high, that the amino nitrogen values are low, and that their sum is usually not equal to the ammonia and amino nitrogen ordinarily found in protein. Hence it may be concluded that hydrolysis is incomplete under the conditions of these experiments. I n general, both forms of nitrogen increase during the progress of the digestion and the maximum is not reached. The permanganate graphs show an entirely different picture. Here the amino nitrogen is almost negligible in quantity and tends to decrease in amount as the digestion
progresses. In the case of the full sample of glue hair where the permanganate color was discharged a t the end of 20 minutes, the amino nitrogen tends to increase, the curve assuming more closely the form of that for the alkaline digestion. With the full sample of cottonseed meal the same condition exists during the middle part of the experiment, but during the last 20 minutes an increased formation of ammonia is accompanied by a decrease in the amino nitrogen. While it is apparent that some of the ammonia in all cases is produced a t the expense of the amino nitrogen, the lack of agreement between the total amounts produced indicates that this is not the whole story, but that the action of the permanganate is more deep-seated, producing ammonia from linkings not ammonified by alkali alone. ACTIONOF PERMANGANATE IN RELATION TO MEASUREMENT OF NITROGENACTIVITY Two fundamental criticisms of the alkaline permanganate method have been made. It has been stated that results cannot be accurately duplicated because of the terminal conditions which deal with an incomplete reaction progressing with considerable rapidity and necessitating a control of conditions not ordinarily possible. It has also been shown that with substances which decolorize the reagent the results obtained are due to quite different causes than those secured with materials which do not decolorize the permanganate solution. The writers have stated in a previous paper3 that the former criticism was of greater theoretical than practical importance. It does, however, become significant, as was stated in a later paper,6 in cases where, because of foaming, the duration of the digestion and distillation is protracted beyond the specified 90 minutes. The curves herewith presented substantiate these statements. 6
J . Assoc. Oficial Agr. Chem., 5, 448 (1922).
90
80 70 60
so 40
25 15
Glue Hair
10
(Full Sample) 5 0 0
IO
20
SO
40
50
FIG. 4
60
70
PIG ti
INDUSTRIAL A N D ENGINEERING CHEMISTRY
November, 1923
1181 90
Y
60
30 25
1,
FIG. 7
0
IO
10
30
40 50 FIG.8
The total quantity of ammonia produced, which is a measure of the activity of the materials under consideration, is the sum of two factors: (1) that formed during the preliminary digestion or (‘solutio”’ period, and ( 2 ) that formed during the measured digestion and distillation periods. The former constitutes most of the ammonia in the first tube i*emoved, while the latter is, of course, the rest of the ammonia collected in the other eleven tubes. The ammonia fraction may be further subdivided into that which is liberated by the alkali alone and that set free by the permanganate. With the exception of the ((basegoods” the values for the first portion vary between 5 and 12 per cent of the total nitrogen. The ((base goods’’ showed 25 per cent ammonia in the first sample taken in the alkali digestion. This material was composed of garbage, beet slop, and leathcr, and had been mixed in the usual way with rock phosphate and sulfuric acid and allowed to stand in a den. The nature of the resulting product is problematical. Had the organic nitrogen been ammonified, it would, of course, have been washed out, as only the water-insoluble portion was used for this work. It is hard to conceive of a degradation product which would be so readily ammonified as this is. With this exception the initial ammonia contents show nothing of’interest or importance. The quantities of ammonia produced up to the taking of the first sample are given in Column 4 of Table I. Column 5 shows the ammonia produced during the remainder of the digestion and distillation. It is a rather interesting fact that, by bringing the temperature up to the digestion point during 15 minutes and holding it there for 5 minutes, results are obtained ‘which, with few exceptions, have the same relative magnitudes as those obtained by the official method, which requires 90 minutes to carry out. Many analysts assume that materials having 50 per cent or more of this
GO
70
l0
20
lo
30
40 50 FIQ. 9
60
70
insoluble nitrogen in the active form are made from base goods of satisfactory quality. If a passing value of 30 per cent, instead of 50 per cent, were used, all the materials would pass by about the same margins that they do in the present official method. TABLEI-ACTION
PERMANGANATE ON ORGANICNITROGENOUS COMPOUNDS (2) (3) (4) (5) 74.35 82.35 70.69 46.58 35.77 68.70 70.00 64.70 46.84 23.16 Full 67.45 62.25 55.75 43.93 18.32 hair Half 6 6 . 6 6 72.00 62.30 39.94 32.06 Chrome uppers 64.35 68.47 42.18 25.29 61.10 Full 52.60 56.95 42.30 32.27 24.68 Cottonseed Half 66.08 62.10 52.18 36.50 25.60 I, full 2 8 . 3 8 33.47 29.19 17.28 ... I, half 4 5 . 6 0 47.15 42.58 22.08 ... 11, full , 2R. 10 23.31 16.40 . 11, half 42.86 38.80 24.91 ... Full 33.79 34.24 29.76 14.43 . .. .. Tartar Pomace Half 4 2 . 0 6 46.69 38.48 26.40 Full 22.68 25.09 20.08 16.36 ... Tobacco stem Half 34.36 31.41 27.70 19.75 ... Full 3 2 . 2 8 , ... Base goods, I Half 5 2 . 6 4 ... 4i:38 .. _ a (1) Active insoluble nitrogen, official method. (2) Active insoluble nitrogen, step method. (3) Active insoluble nitrogen a t 60 minutes. (4) Ammonia formed duriug preliminary digestion plus ammonia present. (6) Ammonia formed after preliminary digestion. OF
MATERIAL Casein Dried blood
{
{
.....
{ {
.. .
.. .. .
...
The influence of the initial ammonification is especially noticeable in the cases of the distinctly low-grade materials, garbage tankage, tartar pomace, and tobacco stems. Although both the slopes of the curves and the amounts of ammonia produced during the solution period are less than those in the high or medium grade, it is the initial ammonification factor that determines the final value. Theimportance of this will be taken up later. The last column shows the ammonia liberated during the regular digestion and distillation periods less the first ’ 80
30
70
70 6b
65
60
50
55
50
$0
45
40
30
35 30
30
25
IO
25
15
/5
Tobocco Stern Sampie x :HalfSample
IO 5 0
/O
20
30
40
50
FIG.10
60
70
25 20
(NH~ only)
0 0
10
20
30
40
50
60
70
FIG. 12
IO
s
INDUXTRIAL A N D ENGINEERING CHEMISTRY
1182
*'
I
I
I
I
Q
I
j
/7
90
ment of s a m p l i n g .
80 There are some differ-
ences in therates, casein and the half sample of i 6 C glue hair showing a c" 4 7 I somewhat more rapid I 5 : evolution of ammonia , than the others I 41 ' y l c throughout the course I o=NH, of the reaction. The d 8 y = ~ ~ . 3 8 -=id7o4 2 differences in the slopes ---=NaOH '' of the curves a t the BaseGoods~ 15 /* (HalfSarnpa/e) lo end of the distillation A b 166' i 4 15 indicate the i m p o r o io 20 30 40 so 60 70 tance of the criticism -F I G . 13 __ previously mentioned -i. e., that a t the end of the process the reaction is still progressing. so rapidly that accurate results are not obtainable. I n the cases of casein, both samples of cottonseed meal, and to a lesser extent the half sample of glue hair, the curves still deviate from the vertical axis, indicating that ammonia is still being given off rather rapidly (from 3 to 7 per cent during the last 10 minutes); while in the other cases the curves practically coincide with the vertical axis, inI
c
(., +
b
r/
7
%6
Ii
TABLE11-PER Sample
1 146 150 162 172 208 389 404 415 419 42.5 __.
465 478 481 487 500 502 508 515 712 717 721B 725 728 731 24862 15 3577 2- 12-2 4-84 1s
5121-46 6-21-2 11-21-7 Peat I Peat 111
1-8-5
70
/
4
CENT
OF ACTIVEINSOLUBLE NITROGEN A S DETERMINED BY
THE OFFICIAL AND MODIFIEDMETHODS
Official Method 1.08
0.42 0.20 0.51 0.30 0.93 0.18 0.30 0.13 0.22 0.20 0.48 0.23 0.36 d. 14 0.43 0.28 0.17 0.52 0.17 0.19 0.41 0.28 0.49 0.31 3.46 3.30 1.23 0.93 2.36 0.51 3.94 3.06 2.19 1.07 0.85 0.79
Modified Method
1.05 0.47 0.21 0.53 0.33 1.02 0.19 0.32 0.14 0.23 0.20 0.55 0.24 0.37 0.16 0.45 0.29 0.17 0.45 0.18
0.20 0.42 0.29 0.48 0.30 3.42 3.38 1.24 0.93 2.25 0.60 4.02 3.41 2.13 1.11 0.76 0.82
Sample
144 148 153 169 182 388 396 412 417 420 455 476 480 483 495 501 505 512 523 716 721A 723 726 730 24860 5-22-1 11 2-1-2 3-10-4 1-5-10 5-21-15 4213 11-21-1 11-21-8 Peat I1
2-1-2
....
Official Method
1.26 0.46 0.54 0.42 0.22 0.11 0.42 0.38 0.81 0.19 0.62 0.61 0.35 0.27 0.40 0.67 0.18 0.40 0.39 0.57 0.24 0.19 0.13 0.48 3.42 0.47 2.93 0.52 1.38 0.18 3.90 0.28 4.52 0.47 0.62 0.57 ..
Modified Method
1.31 0.46 0.61 0.39 0.23 0.12 0.44 0.40 0.77 0.19 0.64 0.64 0.37 0.29 0.42 0.69 0.20 0.43 0.42 0.58 0.22 0.19 0.14 0.49 3.18 0.47 2.82 0.56 1.36 0.21 4.45 0.29 5.10 0.52 0.65 0.60 ..
dicating that less than 1 per cent of the nitrogen is being converted into ammonia during the last 10 minutes. I n some cases, therefore, the reaction evidently has approached completion and little care need be exercised in accurately controlling the final conditions. I n others, however, this is not true. Most of the curves reflect the increase in temperature with the commencement of the distillation, but are fairly
Vol. 15, No, 11
straight above and below this point. The most noticeable exception is the sharp break in the curve for the full sample of cottonseed meal, due probably to the increase in the concentration of the reagents during the last 20 minutes of the distillation. To the second factor-viz., a deficiency in permanganate -Jonese ascribed the anomalous results with cottonseed meal. He also showed that the admixtures of reducing substances with dried blood lowered the activity of this material as estimated by the alkaline permanganate method. This is corroborated by the authors' work. Fig. 2 shows the result of mixing starch with dried blood. These curves were obtained as follows: Dried blood and starch were mixed in such proportions that the dried blood composed 100, 80, 60, 40, and 20 per cent of the mixture. These materials were then handled in the usual manner, the samples being weighed out on the basis of the insoluble nitrogen content. Increasing proportions of starch cause corresponding decreases in the activity of the dried blood until the amount of nitrogen ammonified is reduced from 60 to 20 per cent.' Attention has been called to the fact that most of the ammonia is produced during the early stage of the digestion, and it has been pointed out that the sequence of materials when arranged in order of the percentages of nitrogen ammonified is the same whether based on the ammonia formed during the first 20 minutes or on the whole period of reaction. This suggested the possibility of modifying the present alkaline method so as to reduce the time and attention required. I n previous attempts to accomplish this, stress was laid on the relative amounts of sample and reagent in trying to remedy the above-mentioned defects in the method. The following suggested modification does not do this. The results by it are no more reliable than those obtained by the official procedure and whatever can be said in this respect for or against the one can be likewise said of the other. However, the results by the modified method appear to be just as valuable as those with the present official and they are 0 IO 20 30 40 5 0 . 60 70 obtained with considerFIG.14 able economy of time and trouble. After several trials the following procedure has been selected as giving results comparable to those obtained by the official method: A sample containing 50 mg. water-soluble nitrogen as determined by the official method is weighed into a 500-cc. Kjeldahl flask, the prescribed amount of alkaline permanganate solution added, and the flask connected with a condenser. A small flame under the flask is regulated so that about 100 cc. of distillate passes over in 45 min. The distillate is caught in standard acid and a t the end of this time the excess is titrated. If the solution tends to foam a few drops of octyl alcohol may be added. By this procedure the actual time involved is reduced to one-half of that demanded by the present official method. The attention required is also reduced since the process is a continuous one and not divided into two parts. This method has been compared with the official one on seventy-three different materials. The results by the two methods check as well as do duplicates by the official method. (Table 11) 6
7
Annual Report, Vermont Agr. Expt. Sta., 1899, p. 139. Work done by A. W. Lowell.
