Oct., 1921
THE JOURNAL OF INDUSTRIAL A N D EiVGINEERING CHEMISTRY
condition and can be extracted by salts which are nearly neutral. 2-The colored ferric thiocyanate changes to a colorless compound on the addition of a base, and the change takes place a t a H-ion concentration of pH 5.5, which is thought to be the soil condition when the pink color just leaves. 3-The addition of a few drops of an alcoholic solution of logwood to the potassium thiocyanate extract from the
933
soil indicates by the depth of the blue color the relative amount of aluminium in solution. 4-The limestone requirement by this method corresponds closely to the Veitch method. 5-The method seems to lend itself to the determination of either the acid or basic condition of a soil, and the soluble iron and aluminium found have been helpful in pointing to a source of some of the plant disease troubles.
Studies of the Availability of Organic Nitrogenous Compounds-Part
1‘*2
By C. S.Robinson, 0. B. Winter and E. J. Miller MICEIGANAGRICULTURAL COLLEQEEXPERIMENT STATION, EASTLANSING,M I C ~ I G A N
Several years ago there was started in this laboratory tein present in this raw material. This results in an increase an investigation of the relation between the chemical con- in the soluble nitrogen with the formation of the usual stitution of organic nitrogenous substances and the avail- products of such treatment. Later he made a thorough ability of their nitrogen for plant nutrition. The objects study of the decomposition of dried blood in the soil.‘ He of this work were two in number: (1) to ascertain whether found that over 79 per cent of the nitrogen in this material or not any relation existed between chemical constitution was ammonified during the 240 days of the experiment. and availability, and (2) either to devise a new method or He sums up the processes involved as follows: to improve the present ones for measuring this availability. The ammonia produced during the decomposition of the dried Both of these subjects are of importance because of the blood was derived from (1) the hydrolytic cleavage of the proteins of the dried blood, as evidenced by the rapid vanishing growing tendency to utilize all sorts of organic nitrogenous of the amide compounds from the soil during the first 5 days of materials for fertilizer purposes. the experiment, and ( 2 ) from the decomposition of the products resulting from the hydrolytic cleavage of the proteins. Some RELATIONOF CHEMICAL CONSTITUTION TO AVAILABILITY of the ammonia produced during the first 2 or 3 days, when the The fundamental chemical questions involved in the hydrolysis of the proteins does not seem to have been very may possibly have been due to the de-aminization subject of availability have received considerable attention. extended, of the €-amino group of the lysine in the native proteins of the Jodidi,3 for instance, studied them in connection with the dried blood. With the exception of the amide compounds, decomposition of amino acids and acid amides in the soil. lysine seems to have disappeared most rapidly and completely He found that, of the simple aliphatic a-amino acids, the from the soil. The monoamino acids contributed about 89 per cent of their nitrogen to the formation of ammonia, and short chain glycine was more readily ammonified than the arginine and histidine each contributed about 83 per cent. longer chain leucine and that the cyclic compound phenylIt is apparent from these researches that there is a t least alanine was more resistant to such action than leucine. He confirmed these results by experiments with glutaminic a strong possibility of a relation between chemical constitution acid and tyrosine, and found that amino acid nitrogen is and availability, although but little effort has been made as readily ammonified in the soil as acid amide nitrogen. to correlate this fact with the practical measurement of 13s work was done with simple amino acids, acid amides, availability. The invention of laboratory methods for determining and primary amines. the relative values of organic nitrogenous materials has Kelley4 went a step further and studied the decomposition of various proteins and other complex organic compounds, proved attractive to many investigators, and a number of such as casein, dried blood, soy-bean cake meal, cottonseed such methods have been proposed. Only two, however, meal, linseed meal, zein, and globulin from cottonseed meal. the so-called neutral and alkaline permanganate methods, He found that about 60 per cent of the nitrogen in casein, have shown themselves to be of sufficient value to continue about 50 per cent of that in dried blood and soy-bean cake long in general use. Both of them were developed rather meal, and approximately 30 per cent of that in cottonseed empirically but nevertheless appear to give excellent comparaand linseed meals were easily convertible into ammonia. tive results from a practical viewpoint. THEALKALINEPERMANGANATE METHOD^ In studying the proportion furnished by different groups Much work has been done upon this method by its origihe found that the basic nitrogen WRS the most completely ammonified, with the amide and non-basic fractions variable, nator, C. H. Jones,s who, while appreciating its shortcomings, sometimes one and sometimes the other affordingthe larger has shown conclusively that it is valuable for differentiating in a broad way between high-and low-grade goods. One amount as ammonia. In conclusion he said: It becomes apparent that all portions of the organic nitrogen fault is that it does not give reliable results with vegetable inlthe different materials used as fertilizers and greenlmanures ammoniates like cottonseed meal. This has been attributed are not equally susceptible to ammonification. It is evident, by Jones to a high content of non-nitrogenous organic matter, therefore, that chemical factors inherent in the nitrogen com- a contention which is supported by results obtained with pounds themselves predetermine the availability to some degree. mixtures of high-grade products like dried blood and nonLathrop5; has likewise inrestigated the ammonification nitrogenous materials such as filter paper. Under these of complex organic nitrogenous material. His first work circumstances the availability of the nitrogen in the dried was a study of the effect of “processing” on the organic blood is lowered. nitrogen of fertilizer materials. He showed that such proAnother criticism that has been made4 is that the concessing causes a more or less complete hydrolysis of the pro- ditions must be so carefully controlled that there is difficulty I Received February 25,1921. 2 Published as Journal Article No. 19 from the Chemical Laboratory of the Michigan Agricultural College Experiment Station. Published b y permission of the Director of the Experiment Station. 8 Iowa Agricultural Experiment Station, Research Bullelin, 9 (1912). 4 Hawaii Agricultural Experiment Station, BulEetzn 89 (1915). 5 U. S Department of -4griculture, BulEetbn 158 (1914).
I
Sod Sci., 1 (1916), 509.
Association of Official Agricultural Chemists, “Methods of Analysis,” revised t o November 1919, 1920, 11. 8 Vermont Agricultural Experiment Station, 11th Snnual Report, 1898, 160, 12th Annual Report, 1899, 137; 14th Annual Report, 1901, 219; THI-JOURNAL, 2 (1910), 308, 4 (1912), 438. 4 Street, THISJOURNAL, 2 (1910), 311. 2
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THE JOURNAL OF INDUSTRIAL A N D ENGINEERING CHEMISTRY
Vol. 13,No. 10
in getting concordant results. On strictly theoretical grounds this would be anticipated. In the first place it appears that the reaction which is being measured is an incomplete one and that it is still progressing rapidly a t the time it is stopped. Hence any variation in time will cause 8 corresponding variation in results. It should also be observed that a t the end of the distillation period the contents of the flask consist of the residue from 2.5 g. of KMn04 and 15.0 g. of NaOH in only about 25 cc. of water, giving a, concentration of alkali which is very high and correspondingly effective in speeding up the reaction. In spite of these facts it has been found possible in practice in this laboratory to obtain satisfactory results, although rather careful work is required. As will be shown in a later paper, it seems that in many cases the reaction involved actually does go almost to completion under the prescribed conditions, while in other cases the size of the sample used is such that any ordinary variation in conditions does not permit the evolution of sufficient ammonia to cause material error. The present investigation of the alkaline permanganate method was approached as follows: First, a number of amino acids and acid amides, typical organic nitrogenous compounds of known constitution in which the nitrogen was combined in several ways, were treated with alkaline permanganate in the manner prescribed for the determination of the active insoluble nitrogen. This gave information regarding the action of this reagent on nitrogen in various types of combination. A similar series of experiments was then conducted with some of the better known proteins or substances containing them. Finally a number of samples of commercial base goods were examined: (1) by the alkaline permanganate method for availability, and (2) by the nitrogen partition method of Van Slyke.
Lysine' gave only slightly less than one of its atoms of nitrogen in ammonia form. From the fact that the aamino groups in the other amino acids reacted quantitatively under the conditions of the determination one would probably be safe in assuming that it is this group which is removed from lysine. This is further supported by the fact that the +group reacts only slowly with nitrous acid. That there is a certain parallelism between the sensitiveness of amino groups towards nitrous acid and alkaline permanganate solution is shown in the case of urea. This substance gave up less than half of its nitrogen during t h e period of digestion with alkaline permanganate solution. Complete decomposition of this compound with nitrous acid under the conditions of the Van Slyke method is obtained only after 8 hrs., in comparison with as many minutes for the quantitative removal of a-amino nitrogen from amino acids. The results with asparagine indicate that this conduct is rather typical of acid amides in general, as an analysis of this compound by the permanganate method caused the splitting off of only one atom of nitrogen as ammonia. This presumably was the amino nitrogen, judging from the results with the a-amino acids. Acetamide, on the other hand, having only one atom of nitrogen in its molecule and this in the amide group, gave it all up under these conditions. The ammonification of acid amide nitrogen by alkaline permanganate solution may possibly be a function of the number of carbon atoms in the chain, as Jodidi obtained evidence that this is true with the ammonification of amino acids in soil. The results in this table furnish a basis for predicting the conduct observed with arginine, which yielded only two of its nitrogen atoms as ammonia. From the results with other a-amino compounds we should expect arginine t o give up its a-amino nitrogen easily. We might then anticiACTIONOF ALKALINE PERMANGANATE ON AMINO ACIDS' pate that the other amino group would be split off, but In Table I are given the results of the action of alkaline Kutcher2 has shown that under the influence of permanganate permanganate solution on some of the more common amino the molecule is split into ammonia, carbon dioxide, and acids and other nitrogenous compounds. In the last column guanidinebutyric acid. This last product is then broken are given the number of atoms of nitrogen which are con- up into guanidine and succinic acid. The guanidine is further verted into ammonia. It will be observed that in the case hydrolyzed into ammonia and urea, liberating the second of the monoamino acids with the amino group in the a molecule of ammonia in the process, while the two nitrogen position, i. e., those showing monoamino nitrogen by the atoms in urea are but incompletely ammonified as shorn Van Slyke method, the nitrogen is all converted into ammonia. above with that substance. To ascertain the approximate rate of decomposition of those substances which were not entirely ammonified, the TABLEI-PER CENT OF TOTALNITROGEN,AMMONIFIED WITH PERMANQANATE samples were subjected to further distillation after adding TotalN Available N AtomsN 150 cc. of water. The results are shown in Table 11. Structural in Sub- Percent Percent AmmonMATERIAL Formula Glycine NHrCHrCOOH Glutaminic acid COOH-(CHdrCHNH%COOH Tyrosine OH-CaHrCHrCHNHxCOOH Arginine NHnCNH-NH(CHdr CHNHaCOOH Lysine NHzCHp(CHa)a-CHNHr COOH Diphenylamine CsHs-NH-C6Hs NaphThylamlne CioHrNHz Urea NHz-CO-NHz
stance Substance Total N 18.70 18.61 99.60
ified 1
7.58
7.25
95.80
...
7.74
7.65
98.80
1
32.19
17.42
54.15
2
19.18
7.88
41.07
0.8
8.18
4.93
60.6
0.6
9.76 46.50
4.72 7.50 9.72 9.82 19.81
48.4 16.1 20.9 63.85 90.50
0.5 0.3 0.4
Asparagine COOH-CHrCHNH-CONHz18.22 Acetamide CHsCONHi 21.00
If 0.9
The two amines, diphenylamine and napthylamine, were only partially converted. I Denis 17 .. Biol. Chem., 9 (1911), 365; 10 (19111, 731 studied the action of alkaline permanganate solution on glycine, cystine, alanine and ty-
rosine. He showed that some of the ammonia is oxidized to nitric acid but otherwise all in these compounds goes to ammonia.
TABLE 11-PER
CENT OF
MATERIAL Glycine Glutaminic acid Tyrosine Diphenylamine Naphthylamine Urea
TOTALNITROGEN AXMONIFTSD O N REPEATED DISTILLATIONS -DISTILLATION2nd 3rd 4th 0.00 .... 0.23 0.00 0.00 0.86 0.86 0.80 0.43 0.29 0.31 2.96 2.02 2.00
....
.... .... ....
With the exception of the second moIecule of ammonia from arginine, we are dealing with the simplest conditions possible. If the situation is not compIicated by any such secondary decomposition it may be assumed that the libera1 Zickgrnf [Bet., 35 (1902), 34011 used barium Permanganate to oxidize lysine. He obtained hydrocyanic acid, oxalic acid, and succinic acid as decomposition products, together with a small amount of some substance which he supposed to be glutamic acid. It was isolated m quantity insufficient to identify. In the light of our work his snppasitioa appears t o be erroneous, since the amino group of this compound would probabb have been split off b y the barium permanganate. 8 Benech and Rutscher, 2. B h ~ i d Ckem., . 39 (1901), 278; Kutscher, I b i d . , 32 (1901), 413.
Oct., 1921
THE JOURNAL OF INDUSTRIAL A N D ENGINEERING CHEMISTRY
tion of ammonia is a comparatively simple reaction. The general conclusion which is apparent from the results is that primary aliphatic amino groups are quantitatively ammonified, while amides, cyclic compounds, and amino groups in other than the a positions are ammonified to a less extent. The results obtained by using this method on a complex such as a protein or a commercial ammoniate would beexpected to yidd all of the a-amino nitrogen from the free amino acids, either originally present or formed during the determination, together with a variable fraction of the nitrogen from other nitrogen-containing radicals. In the case of such complexes, however, this variable fraction assumes first place in point of importance. It has been shown that practically all of the free NH2 groups in native proteins consist of e-amino groups of lysine and acid amide radicals of dibasic acids, both of which- are evidently but incompletely ammonified. Consequently the interest shifts to the secondary decompositions or the aminojication stage of the process. There are many data available on the hydrolysis (aminofication) of proteins by acids and alkalies, and we could predict from a priori considerations that this reaction would progress rapidly under the strenuous condition of the permanganate method. We could not necessarily, however, expect i t to go to completion as the total time required for the complete hydrolysis of protein material is several hours, while in the permanganate procedure the reaction is stopped a t the end of 90 min. It is, therefore, an incomplete reaction. But this incomplete reaction is the phase that determines to a large extent the final results obtained, since, as has been shown above, the amino acids resulting from it are quite completely ammonified. This introduces into the method an element of uncertainty which largely determines its value. If the extent to which hydrolysis proceeds approximates the extent of formation of available nitrogen in the soil, the results obtained are indicative of the worth of the material. The further they deviate from this the more inaccurate they become. There is, of course, the added factor of uncertainty in the incomplete ammonification of the other nitrogen radicals indicated above. This, however, is relatively unimportant, since the a-amino nitrogen comprises in the neighborhood of 70 per cent of the total nitrogen of most protein hydrolysates. Were the action of permanganate strictly comparable to that of nitrous acid we should expect, upon complete hydrolysis, the ammonification by the permanganate of all of the monoamino nitrogen, one-half of the lysine nitrogen, one-third of the histidine nitrogen, one-fourth of the arginine nitrogen, and an uncertain fraction of the amide nitrogen. Kutcher’s results, as well as our own, show that a secondary reaction ammonifies an additional atom of nitrogen from arginine and, with a reagent such as the permanganate solution used, such secondary reactions undoubtedly take place with other compounds as well. This makes possible only a general analogy between the effects of the permanganate solution and a reagent like nitrous acid which is fairly specific in its action. However, it is apparent that a similarity does exist in the case of several of the common proteins which were investigated with this point in view.
936
sum of both the amino and acid amide fractions is given in the last column of Table 111 for comparison with the active insoluble nitrogen. The figures show that except in the case of gelatin the permanganate digestion causes the ammonification of a portion of the nitrogen greater than that represented by the total amino but less than that equal to the sum of the amino and acid amide fractions. TOTALNITROQEN OF PROTEINS IN VARIOUSFORMS Active Amido Total Insoluble Amido Amino Amino Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Casein 13.83 73.25 10.82 69.00 79.82 Egg albumin 12.64 63.35 8.65 59.26 67.91 Glutin 13.60 70.30 21.48 57.52 79.00 Gelatin 15.15 62.00 1.48 59.18 60.66 Fibrin 14.76 75.15 8.52 69.53 78.05 Albuminfromblood 11.81 71.20 7.54 66.81 74.35 TABLE111-PER
CSNT OF
+
ACTIONOF ALKALINE PERMANGANATE ON COMMERCIAL NITROGENOUS MATERIALS It remained now to test out the above results with commercial materials. The total amide, mono-, and diamino acid nitrogen were determined after hydrolysis with acid, and the usual procedures were carried out in the estimation of available nitrogen by the alkaline permanganate method. TABLEIV-PER
TOTALNITROGEN O F FERTILl!ZERS IN VARIOUS FORMS Soluble Nitrogen (“3 Amide Total %:t?$ Amide Amino Amino Nitrogen Insoluble Nitrogen Nitrogen Nitrogen 2.76 40.21 20.65 39.14 59.79 3.49 42.40 13.46 39.53 52.99 CENT O F
+-
Peat (dried) Peat (wet) Pulverized sheep manure 2.34 Hay and silage 3.10 Fruit and vegetable 3.74 Bone meal 2.48 Animal tankage 4.40 Pure bone meal 3.12 Castorbeanpomace 4.78 Cottonseedmeal 6.73 Kazoo brand 2.32 Mixed-chromeuppers 9.88 Bone 3.05 Glue hair 8.49 Beef scraps 8.72 Animal tankage, I1 4.62 Hair wlste 14.07 Dried blood 14.01
28.20 16.71 43.85 46.43 56.94 62.81 48.96 50.51 66.81 51.22 67.21 64.55 74.54 59.30 70.85 71.31
+
14.53 6.13 5.09 4.84 7.95 8.01 11.09 10.33 6.17 4.76 5.24 5.89 5.96 5.84 9.52 6.56
41.88 42.22 49.20 49.20 54.31 55.76 55.85 58.09 58.18 60.32 60.98 66.77 68.11 69.26 70.00 75.23
56.41 48.35 54.29 54.04 62.26 63.77 66.94 68.42 63.35 65.08 66.22 72.66 74.07 75.10 79.62 81.79
’
Of the fractions obtained by the permanganate method the water-soluble fraction should consist of the ammonia and nitrate nitrogen, together with such soluble organic compounds as may be present, such as free amino acids,
ACTIONOF ALKALINEPERMANGANATE ON PROTEINS Samples were digested with alkaline permanganate solution according to the official method, assuming all of the nitrogen to be insoluble. That portion ammonified was classed as active insoluble. Other samples were hydrolyzed with acid in the usual manner and the monoamino, basic or diamino and acid amide nitrogen determined. The basic amino fraction consists of one-fourth of the arginine, one-half of the lysine, and one-third of the histidine nitrogen. The
acid amides, urea, etc. The active insoluble portion should include that portion of the protein material which is hydrolyzed and ammonified in the course of the digestion. From the results with individual proteins and simpler compounds we should expect that the sum of the active insoluble and
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THE JOURNAL OF INDUSTRIAL A N D ENGINEERING CHEMISTRY
water-soluble portions minus the nitrate and ammonia nitrogen would show the same relation to the amino and amide fractions as did the active insoluble portions of pure proteins. Table IV gives these figures, which are shown graphically in the figure, the values being plotted. in order of ascending values of the amino nitrogen. The similarity in trend is very apparent. It could not, of course, be expected that the results would agree since, for reasons already pointed out, the active insoluble portion includes only part of the amino nitrogen and a variable fraction of the acid amide nitrogen. Evidently the results with these materials do not show the same consistency as did those with pure proteins and the simpler compounds. I n 50 per cent of the cases, hydrolysis was apparently incomplete, assuming that all amino nitrogen liberated during acid digestion is capable of being ammonified in the permanganate digestion. This is shown by the fact that the ammonia produced in the process was not equal in amount to the total amino nitrogen. I n the other cases results analogous to those with proteins were obtained, i. e., apparently all of the amino and part of the amide nitrogen was ammonified. These results are, nevertheless, in harmony with the considerations stated above, it having been pointed out that the reaction is still incomplete a t the end of the digestion period in the permanganate method.
DISCUSSION AND SUMMARY In the light of the discussion in the earlier part of the paper, together with the experimental data, certain general statements regarding the availability of organic nitrogenous
Vol. 13, KO. 10
compounds seem justified. It is, of course, conceded that inorganic compounds of ammonia and nitric acid constitute a class of immediately available material. Assuming that the ammonifying, as distinguished from the aminojying or hydrolyzing, power of the alkaline permanganate solution is comparable to the action of soil agents, we may add to the nitrogen of the above members of the available class of compounds all amino nitrogen present in the form of aamino acids and a portion of that nitrogen present as acid amides. Then there is another class of compounds which we have called the potentially available class. It includes such substances as may be converted into members of the former class, and consists of a portion of the acid amides, the peptides, which can be hydrolyzed to amino acids, and primary and secondary amines. The peptides probably form the great bulk of this class so far as ordinary fertilizer maaterials are concerned. This class is the uncertain quantity in evaluating any material from the fertilizer standpoint. In some cases transformation into the available class is so easy and complete that there can be no practical distinction between the two. In other cases this process is so slow that the' other extreme, i. e., the unavailable class, is approached. Fundamentally, the problem of devising a method for the determination of the availability of organic nitrogen compounds is the invention of one which will properly estimate the rate of aminofication of the members of this class. Up to the present the permanganate methods have proved the most satisfactory. They are being studied further in the light of the work reported in this paper and the results will be published in the near future.
The Chemical Constitution of Soda and Sulfate Pulps from Coniferous Woods and Their Bleaching Qualities' By Sidney D. Wells MADISON,WISCONSIN U. S. DEPARTMENT OF AGRICULTURE, FORESTSERVICE,FOREST PRODUCTS LABORATORY,
In looking for a solution for the periodical shortage of raw materials for paper manufacturing, the investigator soon realizes the existence of the large and widespread supplies of wood too resinous for use in the sulfite process, and too resinous, dark colored, or hard for the manufacture of satisfactory mechanical pulp. Of all our standing timber only 3 per cent, existing in the Lake and Northeastern states, is available for use as a source of supply for the manufacture of over 70 per cent of our wood pulp production. In the same areas and in the southern states within a reasonable distance of the important publishing centers, occur stands of various species of pine, tamarack, and other coniferous woods amounting to 20 per cent of the total stand in the United States. They possess a very satisfactory fiber, but can be economically pulped only by alkaline processes, such as the soda or sulfate, by which the resinous matter is readily rendered soluble. Unfortunately, soda or sulfate pulps from coniferous woods are difficult to bleach t o a satisfactory degree of white, and only relatively small amounts are produced for special purposes. Even spruce, which is used in large quantities in the manufacture of bleached pulps by the sulfite process, yields a soda or sulfate pulp about as difficult to bleach as longleaf pine or tamarack. Many investigators have attributed this to the presence of impurities such as lignin, but the writer has long felt that it is due to coloring matter present in the wood or produced during digestion, which is very small in amount but of high tinctorial power. In fact, it has been 1 Presented
before the Section of Cellulose Chemistry at the Blst Meeting of the American Chemical Society, Rochester, N.Y.,April 26 to 29, 1921.
felt that sulfate pulps from the pines, spruces, and firs (even though cooked for strength and dark brown in color) more nearly approach cotton cellulose in chemical or physicd characteristics than well-cooked sulfite pulps from the same wood. Studies were therefore undertaken to ascertain the chemical characteristics of typical soda and sulfate pulps from white spruce which were cooked under carefully regulated and known conditions, and also of portions of the same pulps bleached to various degrees.
PREPARATION OF PULPS The pulps were cooked in the semicommercial tumbling digester in use a t the Forest Products Laboratory which has a capacity of 100 lbs. of dry pine chips, or a production of from 40 to 50 lbs. of bone-dry pulp per charge.' In Table I are presented the cooking conditions and yields of pulp obtained. The bleached pulps were prepared from the unbleached pulp described above by bleaching with definite amounts of bleaching powder solution a t 100" F. in glass vessels provided with mechanical stirrers. The bleaching operation in each case was continued until just a trace of active chlorine remained, as shown by starch iodide indicator. The pulps were then washed free from bleach residues, pressed, and allowed to dry by exposure to the air a t 70" F. They were then stored in airtight fruit jars until needed. The amounts IKress, Wells and Edwardes, "The Equipment and Operation of an Experimental Pulp and Paper Laboratory," Paper, a6, No. 13 (lglg), 11; Pulp Paper Mag. Can., 18, No. 23, 588.
