THE ISOLATION OF PICOLINE CAR: 3 X Y L I C ACID.
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souring but favored the development of an oR'cr:site, putrid odor, and a bitter taste. This effect was much less noticeal>li.in the samples pasteurized a t 65' than in those of the same origin pasteurized a t 8 5 O , the same precautions having been taken to prevent subsequent contaniination in either case. NEW Y O R K
CITY,
June, 1908.
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THE ISOLATION O F PICOLINE CARBOXYLIC ACID FROM SOILS AND ITS RELATION TO SOIL FERTILITY.' BY OSWALDSCHREINER AND
EDMUND C
SHORES
Received June 4 1908.
Recent investigations have shown, contrary to the generally accepted belief since Liebig's time, that lack of productivity in soils is not always due to the lack of nutrient substances, but even with an abundance of these, lack of productivity may he produced by the presence of substances actually deleterious to plant growth. The presence of toxic substances has been virtually established by the properties which the soil and soil extracts possess. When an unproductive soil is extracted with water, the aqueous solution has been found to possess the properties of the soil itself so far as plant growth is concerned. When a good soil is extracted, a good soil extract is obtained, and a poor soil yields a poor soil extract, although the extract may have been made from adjoining fields of the same soil type and be practically identical in mineral composition. Such poor soil extracts produce in many instances plants which are far less developed than similar plants grown in pure distilled water under identical conditions. I t has been found that these toxic properties can be removed from such a soil extract by a number of quite simple operations, such as treatment with carbon black, ferric hydroxide, and other absorbent agents, whereby the toxic material is apparently removed from the solution. This action seems to be quite common for all toxic soils studied although in other respects the properties of toxic soil extracts differ appreciably. In some cases the toxicity can be destroyed by merely boiling the extract and this is due in some cases to a destruction and in others to a volatilization. In the latter case the toxic properties can be found in the distillate. The toxicity can also be much weakened by dilution with pure distilled water so that in the weaker solution there may even result a stimulation in growth such as is commonly observed with a large number of poisons in weak solutions. These toxic properties are also overcome in many instances by the addition of comparatively minute amounts of pyrogallol, naphthylamine, and other compounds, a phenomenon which can be explained only by assuming that these compounds have acted upon the toxic substance, or have so altered the plant by stimulation Published by permission of the Secretary of Agriculture.
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OSWALD SCHREIUER 4 N D ED3II'UD
C . SHORES.
or otherwise as to overconie t h e m 'I'hat mineral salts siicti as sodiiini nitrate or calcium cnrlmnate also aid i i i the tiest ruction of tosic substances has already been showti in a previous p i p e t . ' I t is of course well I\iio~riithat toxic soil cnnditions caii be brought about by escess of salts nr t tic 1)t(wiicc of unfavorable inorganic conipounds, such as ferrous iroii, ctc., h i t thc properties of the tosic soil solutions that h a w bceii esaiiiined i i i these lahoratorirs point strongly to the fact that the toxic sul,stxiices itrc orgaiiic antl that tlie productivity of a soil depends largely upon tlic condition of the orgaiiic tnatter i n the soil and the processes wliich arc a t work iii dcstroyitig tlie plant remains. The review just given has s l i o ~ vthat the toxic properties of soils have been demonstrated and that the esisteiicc of toxic substances is a reality with which it is necessary to (leal iii future soil studies on the fertility and infertility of our agricultural lantls. .I \.cry iniportant phasc of u ork in these soil studies reniainetl to he clone before a ttioroiigh knowledge of these suhstaiices antl tlie best IIIBLLIISof treatment i n practice could be attained. >Ian?- of their properties it has Ixeii shon-11 hat1 alrcacly been determined in so far as they related to plant gron-tli. 9 o t m l y had their physical properties hevii stu(lied. h i t tlir pliysiological properties, in their effect oii tlic iiutritioii of plants 0 1 ntlier physiological functions had also been ascertained for iii;iiiy soil. one way t o attain this knowledge is to isolate these sub stances froni soils and study their properties. I he problem of isolating organic su1)stnnces from soils has been attempted from time to time in the history of agricultural science. 13racoiinot,' W a l s e ~G, ~~ d eand , ~others eveii attempted to show by chemical means the presence of the toxic suhsiances and i t is largely due to their failure to obtain positive reactions that the matter was dropped for many years. Investigations have been reported from time to time on the isolation of various humus acids, ulmic, geic, crenic, apocrenic, etc., but the composition of the acid isolated has been a most variable one differing with each investigator and with each method employed so that i t would not be far from the truth to say t h a t none of these acids were shown t o exist and that me know almost nothing ahout their chemical conipositioii or constitution. The work of isolatitig and stiirlying the nature of such conipounds >,
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T H E ISOLATION OF PICOLINE CARBOXYLIC ACID.
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is necessarily slow and difficult to accomplish. In the first place, the amount may be very small and the composition of the organic matter of the soil is undoubtedly very complex, being made up of large numbers of organic chemical individuals. The absorption of the soil itself for organic substances is very great so that the ordinary solvents such as water, alcohol, chloroform, etc., take up but very minute amounts of organic material. Then, too, there is an astounding lack of method for handling problems of this kind as there is no previous work which can serve in any measure as a guide. As pointed out above, it is questionable whether any pure organic compound has ever been isolated from a soil and identified until very recently, although it is obvious that a large number of organic compounds must exist. The amount of organic matter in ordinary soils is really very large, the average content being as high as 2.06 per cent. for the soil and 0.83 per cent. for the subsoil, determined on thousands of samples of soil and subsoil from all parts of the United States covering in all 237 types of soil. It is obvious that this organic matter is of very complex composition as its properties are quite different in soils from different localities. The amount of nitrogen also is considerable, as much as 0.1to 1.0 per cent., and when we consider that only a small amount of this nitrogen is in the form of ammonia or nitrate and that the larger amount is in organic form, it is obvious that the problem becomes an important and very complex one. It will be apparent to any one who realizes the paucity of knowledge of the organic matter in soils that the isolation of any definite organic compounds from the soil is not any easy task. When this is narrowed down to the isolation of toxic compounds the difficulties are greatly increased. Before outlining the methods by which this problem has been approached some of these difficulties should be considered for the matter resolves itself into a choice of methods to surmount these difficulties. The water extract of an infertile soil usually contains but a very small quantity of organic matter, and it is a reasonable assumption that the toxic organic matter is only a part of the total organic matter present. Such being the case it is evident that the isolation of any organic compound from a water extract of a soil in sufficient amount to establish its identity and study its properties is in most cases a problem that does not come within the limitations of laboratory work. I n other words, the amount of soil or soil extract necessary to handle would be too great for ordinary laboratory appliances and methods. This difficulty due to the small quantity of organic toxic material which must be present in the soil solution is much increased by the fact that the toxic substances are unknown compounds with unknown properties. The only certainty in modern toxicology in the detection of poisons in mixtures of organic matter such
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THE ISOLATION OF PICOLINE CARBOSYLIC ACID.
1303
of pyruvic acid. When the neutral soil extract is treated with these reagents there is produced a very slight greenish color which, since the soil extract is of an orange color, might be expected in the presence of small amounts of pyruvic acid. The delicacy of the reaction was tested by adding known quantities of pyruvic acid to the neutral soil extract and adding the reagents and it was found t h a t the reaction was not a t all delicate in the presence of the other material present in the soil extract, as much as 500 parts per million being required to give the characteristic blue color, lower concentrations producing greenish shades. These reactions indicate a t least the probable presence of pyruvic acid as well as other compounds not yet isolated. The possible presence of this acid in the soil is of interest for i t may be that through the agency of ammonia in the soil the change from the open chain non-nitrogenous pyruvic acid, to the ring form nitrogenous uvitonic acid may be effected as is done in the laboratory with alcoholic ammonia. Uvitonic acid has been sought for in the various soil extracts, but so far with no indication that it is present. When picoline carboxylic acid is prepared from the neutral soil extract by the method stated above there is separated from the crystals a dark colored mother liquor containing nitrogen. When this is evaporated nearly to dryness there is usually a further separation of a small quantity of picoline carboxylic acid but no other crystalline substance has been obtained or observed. The mother liquor a t this stage, however, is thick and viscous and of such a nature that crystallization of any substance present would be difficult. When the mother liquor from which picoline carboxylic acid has been separated is made alkaline with sodium carbonate and treated with a solution of diazo sulphanilic acid a dark red color is slowly formed. The color produced by this reagent with certain organic compounds was first noticed by Ehrlich' and further described by Pauly,2 who found that it could be used for the detection of histidine. Histidine gives with this reagent a dark red color, which remains red on dilution and no other protein decomposition product except tyrosine gives this color, all others giving yellow colors, which disappear on dilution. According to Pauly, the distinctive color given by histidine and diazo sulphanilic acid is due to the imidoazole group, histidine being u-amino P-imidoazole propionic acid. The color obtained by treating this material with diazo sulphanilic acid does not appear to be that obtained with histidine, being slower in appearing and soon losing the red tint on dilution. The same color can be obtained in the distillate on distilling the original soil with steam and is much more pronounced if the soil be made slightly alkaline before distillation. It has been found 2 kliit. .lIt.d., 5 , 285 (1882). 2. p h y s h ~ l .Chem., 42, 508 (1904); 44, 159 (1905).
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