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tained, rather t h a n t o first convert these into percent- of t h e filtrate remaining from t h e 2 0 cc. used for t h e ages of nitrogen, a n d t h e n t o make t h e comparison. above determination is permitted t o s t a n d a t room A big saving in time a n d labor is t h u s effected. temperature for a given time, preferably over night T h e manner in which I operate is as follows: I for a period of 16 hours, after which time a second prepare a cold water extract of malt with distilled water acid ratio determination is made. Malts showing t h e a t room temperature in t h e proportion of I p a r t of greatest increase i n t h e acid ratio are those having t h e malt t o 3 of water, using 5 0 grams of malt finely greatest peptic strength. This increase :n t h e acid ground. T h e water a n d t h e malt are shaken at room ratio m a y amount t o from j t o 30 or even more. temperature for a period of 30 minutes b y means of T h e reagents employed in t h e above determinations some convenient shaking device, a n d t h e n promptly aside from t h e t e n t h normal alkali found in every Iaborafiltered into a clean, d r y flask. About I O cc. are first tory are prepared as follows: permitted t o pass through t h e filter a n d are t h e n 0.5 p e r ceizt solzrtioii of p h e u o l p h t h a l e i n in soyGalcohol returned t o t h e filter again, this procedure being re- t o which is added a sufficient amount of t e n t h normal peated a second time. Exactly 4 j minutes after t h e soda t o impart t o i t a slightly pink color. t i m e of starting t h e extraction or 1 5 minutes after re40 p e r c e n t j o r m a l d e i i y d e solzilioii (technical strength) moving t h e solution from t h e shaking machine, a por- t o which is added j per cent by volume of t h e above tion of t h e filtrate is titrated in t h e following manner: phenolphthalein solution a n d a sufficicnt amount of T w e n t y cc. of t h e solution are placed in a beaker of j o o t e n t h normal soda t o impart t o it a slight b u t decc. capacity a n d about 2 0 cc. of distilled water a n d 0 . j cc. cided pink color. In conclusion, I wish t o point out t h a t t h e acid ratio of standard phenolphthalein solution are t h e n added, method developed b y me can be used t o advantage whereupon t h e mixture is titrated with T h e number of cc. of t h e .!\'/IO sodium hydrate re- in determining whether a certain malt has been prequired t o produce a faint pink coloration are noted. pared from a specific t y p e of barley or whether i t repreT h i s titration represents t h e natural acidity a n d can, sents a mixture. Aside from its great simplicity it if so desired, be figured over t o a percentage basis a n d has t h e further ad\-antage t h a t one single determination b y multiplication with t h e factor 0.009 be stated in suffices for t h e estimation of both t h e preformed amino acids present in t h e malt a n d t h e proteolytic strength, terms of lactic acid. However, this is not necessary, inasmuch as t h e titration figures themselves serve a n d furthermore, t h a t all of t h e work can be carried out a t room temperature and will yield reliable resrilts equally well for purposes of comparison. Ten cc. of without requiring special sliill and a degree of accuracy t h e standard formaldehyde are next added a n d owing t o t h e reaction Kith amino groups the mixture becomes which, owing t o t h e time required, would render the strongly acid. T h e titration with ~ V / I O hydrate is method unsuitable for technical work. t h e n continued until t h e solution has assumed a deC H E M I C A L LABORATORY, THE \VX. R A I I R SOKS' C O X I ' A N Y ~IAKITOW \Vrscossrs OC, cided pink color. The additional amount of acid necessary t o neutralize this increased acidity gives t h e measure of t h e amino groups which have entered into A MODIFIED METHOD FOR DETERMINING CARBONFREE ASH I N PLANT SUBSTANCES t h e reaction. For brewing purposes a malt having t h e By GEOKGBE. BOI.TZ greatest amount of amino groups is t o be preferred, b u t Received May 7 , 191.5 only provided t h e original acidity has been fairly In t h e usual method for determinirg ash in plant high, a n d t h e relation or ratio between t h e formol acidity a n d t h e natural acidity, obtained b y dividing substances, where no pro\-ision is made for purifying t h e number of cc. representing t h e amino acids by t h e t h e ash, the error due t o t h e presence of carbon dinumber of cc. representing t h e natural acidity, is as oxide in combination with bases present is, in many I : 1.00 or greater. This ratio which I designate as cases, quite large. T h e percentage of error is in prot h e a c i d r a t i o o,f m a l t is a n e n t i r e l y new ,factor in malt portion t o t h e amount of basic material t h a t is free valuntioiz. A really good malt should be high in original t o unite with carbon dioxide during combustion. acidity a n d should shon- a n acid ratio of about I : 1.10, Ash containing large amounts of calcium, magnesium which for convenience sake I express simply as 110. a n d potassium should he corrected for t h e carbon diThere are some very good malts, especially low dried oxide i t contains, mhile in most determinations of ash distillers' malts, which will have a n acid ratio as'high a correction for unburned carbon, a n d in some cases, as 130, although I ha\-e failed t o find a n y having a a correction for sand, is necessary. -4 procedure which has proved t o be very satisfcichigher ratio. Large berried Western barleys (Bay Brewing, White Club, etc.) yield malts having a low tory in determining t h e real ash in plant substances acid ratio (about 88) ; two-rowed barley malts have a n is as follows: Veigh from z t o I O grams, depending upon t h e maacid ratio of a b o u t 90 t o 100,a n d regular six-rowed malts prepared from Manchuria barley about I I O t o terial, into a platinum dish. Ignite over a low flame until most of t h e carbon is burned off. Cool, cover 120. It is quite likely, however, t h a t these standards for t h e different types of malts are subject t o change, t h e dish with a watch glass a n d add through t h e lip like all other malt characteristics, from season t o of t h e dish about 2 0 cc. of hot distilled viater. Filter into a weighed zoo cc. Erlenmeyer flask, wash season. I n order t o obtain comparative figures as t o t h e t h e residue 3 or 4 times xvith hot water, replace t h e proteolytic strength of different malts, t h e balance filter paper 1%-ithresidue in t h e platinum dish. dry ~
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a n d ignite until practically all t h e carbon is consumed. Transfer t h e , remaining ash t o t h e Erlenmeyer flask with hot water, using a policeman t o loosen a n y particles t h a t m a y adhere t o t h e dish. Evaporate t h e moisture a n d d r y t h e ash at 110' C. until thoroughly d r y ; weigh. T h e weight minus t h e weight of t h e flask represents t h e crude ash. Connect t h e flask containing t h e crude ash t o a n apparatus' for determining t h e carbon dioxide, t r e a t t h e contents of t h e flask with 80 cc. of distilled water free from carbonates a n d 2 0 cc. of,dilute hydrochloric acid ( I : I O ) . Aspirate purified air through t h e appar a t u s while liberating t h e carbon dioxide. Boil for 30 minutes a n d absorb t h e gas in 5 0 cc. of a 4 per cent solution of sodium hydroxide. Drain t h e sodium h y droxide solution o u t of t h e absorption tower a n d wash t h e remaining caustic solution out of t h e tower with 2 5 0 cc. of Con-free water. Exactly neutralize with normal hydrochloric acid! using phenolphthalein .as indicator. Add 2 drops of methyl orange solution ( I gram in 1000 cc.) a n d titrate with N / 2 0 hydrochloric acid until t h e color of t h e methyl orange is just changed. F r o m t h e number of cc. of N / 2 0 hydrochloric acid used subtract blank: I cc. N / z o hydrochloric acid = 0 . 0 0 2 2 gram carbon dioxide. T h e titration where phenolphthalein is used is ignored. T h e carbon, sand a n d silica are determined a s outlined on page 2 2 in Bzilletiiz 107, U. s. Bureau of CWemistry. T h e carbon dioxide plus t h e unburned carbon a n d sand is subtracted from t h e weight of crude ash. The remainder represents , t h e amount of carbon-free ash.
of amino acids from soils indicate . t h a t t h e proteins are decomposed in a soil in mucfi t h e same way as in acid hydrolysis or in animal digestion. If this view is correct a n d t h e soil protein is hydrolyzed as in digestion b y acids or enzymes we should expect t o find in soils all of t h e intermediate products of protein hydrolysis provided t h e methods are sufficiently accurate for their identification. U p t o t h e present time, however, only a few of t h e primary cleavage products have been found. Although t h e complex, first stage, decomposition products of t h e proteins have not been found t o occur or persist in soils, their presence has been anticipated. It is t h e object of this paper t o report t h e presence of certain bodies, presumably proteoses and peptones! resulting either from partial hydrolysis of proteins or b y t h e synthetic action of microorganisms. T h e present methods for differentiating t h e rarious proteoses a n d peptones are unsatisfactory. These compounds are classified according t o their varying solubilities, especially in ammonium sulfate solutions of different concentrations. T h e differences in composition betureen t h e various members of t h e respective groups remain t o be more accurately established. Because of t h e chem'cal similarity of these substances, t h e difficulty attending their separation, a n d t h e uncertainties in their identification, pure proteoses a n d peptones are not easy t o procure, a n d in a n investigation of this character where t h e y are present in soil in comparatively minute quantities, only qualitative reactions can be employed t o show their existence. Oriro AGRICULTCRAL EXPERIMEXT STATION Xumerous investigations' on t h e hydrolysis of proWOOSTER.OHIO teins hal-e shown t h a t t h e protein molecule is gradually broken down into a series of long chains of THE PRESENCE OF PROTEOSES AND PEPTONES I N amino acids. These chains are known as proteoses, sons peptones, a n d peptides according t o t h e number of B y E. H. TVALTERS units in t h e chain. These still possess t r u e protein Received a p r i l 14, 1915 characteristics. Further hydrolysis causes t h e ultiRecent investigators concur in stating t h a t t h e chemistry of soil nitrogen is essentially t h e chemistry mate spIitting of these protein-like substances into of protein undergoing hydrolysis. Proteins find their amino acids of knon-n chemical structure. It n-ould be beyond t h e scope of this paper t o dwell way into t h e soil in t h e form of plant a n d animal upon t h e various proteoses a n d peptones which are dCbris, manures a n d fertilizers. ,4s soon as these described in detail in t h e literature. Only their substances are incorporated in t h e soil, hydrolytic general characteristic properties will be given. decompositions ensue. T h e investigations of Shorey,2 J ~ d i d i ,R~~ b i n s o n , ~ T h e proteoses have been considered as t h e interSuzuki.: Lathrop a n d Bron-n:6 a n d Kelley: on t h e mediate substances in t h e peptonization of proteins classification of t h e nitrogenous decomposition prod- whose neutral or faintly acid solutions do not coagulate ucts as ammonia, monoamino acids, diamino acids, on boiling. They are subdiT-ided into primary and etc., a n d t h e work of Schreiner a n d Shorey.s Shorey,9 secondary proteoses, T h e primary bodies possess Lathrop,'O a n d Robinson," on t h e isolation of a numbcr a higher molecular weight a n d greatly resemble t h e proteins; they are precipitated and t h u s separated 1 THISJOUR,NAL, 4 (1912). 611. from t h e secondary proteoses by either completely 2 E. C. Shorey, U. S. D. 4 . Hawaii Sta., A n x . R e p ; . , 1905, pp. 25-38 saturating their aqueous solutions with sodium chloride a n d 1906, pp. 37-59. 3 S. I,. Jodidi, hlichigan Sta., Tech. Bull., 4 (1909); Iowa Sta., Research or half saturating t h e m with ammonium sulfate. B u l k . I a n d I11 (1911). According t o Pick2 and Haslam3 t h e primary pro4 C. S. Robinson. Michigan Sta., Tech. Bull. 7 (1911). S. Suzuki, Bull. Coll. T o k i o , 7 (1907), 513. teoses are represented b y a t least three substances: 8 E . C. Lathrop a n d B. E. Brown, T H r s JOCRNAL, 3 (1911), 6 5 i . hetero-proteose, a-proto-proteose, a n d P-proto-proteose. 7 W. P. Kelley. J . A m . Chem. Soc., 36 (1914). 429-444. ~
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a Oswald Schreiner a n d E. C . Shorey, C . S. Dept. of Agr., Bureau of Soils, Btrll. 74 (1910). 9 E. C. Shorey, U. S . Dept. of Agr., Bureau of Soils, Bull 88 (1913). 10 E. C. Lathrop, J . Am. C h e m Soc., 34 (1911), 1260. 1's. C. Robinson, I b i d . , 33 (1911), 564.
1 For general references see E. Abderhalden, "Uioqhemisches H a n d lexikon," Vol, I V ; "Handbuch der Biochemischen Arbeitsmethoden," Vol. I1 and 111; Gustav h l a n n , "Chemistry of t h e Proteids." 2 E. P. Pick, Ztschr. physiol. Chem., 24 (189i), 246. 1 H . C. Haslam, J . Physiol., 32 (1905). 2 6 2 .
