IO24
T H E J O L - R S A L OF I-YDCSTRIAL A S D ESGISEERIilrG C H E M I S T R Y
for f a t present) and it is probable that t h e fat could be determined on t h e dried precipitate b y powdering and extracting with ether in a Soxhlet apparatus, similar t o a method proposed b y A , E. Paul. Milk sugar (lactose) can be determined directly (by copper reduction gravimetric or by volumetric methods) on the filtrate before inversion, so we have here an outline for a “complete” analysis of sweetened condensed milk, using a t t h e most but three aliquots of the original diluted sample. The use of copper sulfate as a precipitant of proteins and mill K . \ X K L I S STRKET, S S W Y O R K C I T Y
ESTIMATION OF SUGAR IN MEAT PRODUCTS, PARTICULARLY EXTRACTS’ By \V.
R. SXITH
Received July 1 2 , 1916
The best precipitants for t h e nitrogenous bodies in meats and meat extracts which interfere with t h e reduction of Fehling’s solution by sugar, are mercuric nitrate, mercuric acetate, and picric acid, followed b y phosphotungstic acid, with or without hydrochloric acid. For polarimetric x o r k t h e excess of mercury does not have t o be removed, b u t for copper reduction this is absolutely necessary. Irrespective of other qualities this fact makes t h e use of mercuric salts tedious and difficult. The removal of t h e excess of mercury is troublesome; although it has been performed with ease under certain conditions, which appear t o be distinct acidity, sufficient rapidity of the current of MzS, and not t o o concentrated solution, several chemists have found difficulty with it, especially when meat extracts containing high percentages of sodium chloride are being tested. If this p a r t of the clarification is not well done, t h e succeeding steps cannot be carried out successfully. This criticism applies particularly t o t h e nitrate of mercury; not only is t h e acetate of mercury more efficient as a precipitant, b u t the nitric acid greatly hinders the furmation of mercuric sulfide. For these reasons picric acid, when used in place of mercuric salts, not only- is easier of manipulation, but saves a very great proportion of the time required. Folin states,? “in t h e presence of copper, alkaline picrate solutions are not reduced b y sugar,” and this has been confirmed b y the experience of t h e writer. The sugar can, moreover, be estimated b y t h e picramic 1
2
Published by permission of the Secretary of Agriculture. J . A i d Chem., 22 ( I O I S ) , 327-9.
T‘ol. 8,K O . T I
acid reaction recently published by Benedict,’ Bernhard,2 and Myers EST13IATIOK O F S U G A R
I n this work the sugar was determined by reduction of Fehling’s solution and estimation of the copper in the precipitate by L o r ’ s iodide method. As described in a former paper,* difficulty is sometimes experienced (with t h e mercuric salts filtrate) i n filtering off the cuprous oxide, which comes down yellowand more or less soluble in water. Less difficulty has been found when picric acid is used The NunsonWalker method usually gives good results, especially when the percentage of sugar is high. Out of I j o tests of various methods the following are representative: PER CENT SCGAU FOUKD BY Munson-Walker Bertrand Cured M e a t E x t r a c t . , . . . . . , . , , , , . , , , . . . . . 4 . 2 i 4.31 Bouillon Cubes.. . , . . , ~, . . . , , . , , , , , , , , , . , 1 . 7 ; 1.88 Extract from Spleens and Livers.. , , , , . , , . , I . 81 1 82
PRODUCT
.
There are indications however, t h a t Bertrand’s method is more tolerant of impurities t h a n t h e MunsonWalker. The following tests were made on a single solution, clarified b y the picric acid method From extracts of beef: Percentages Sugar b y Bertrand method.. . , . . . , . . . . . , . , , . . . . . . . 0 . 0 9 0.13 0.39 Sugar by Munson-Walker: using 50 cc. of sugar solution 0 . 2 8 using 25 cc. sugar solution and 25 cc. w a t e r . , , . , , 0.20
The results b y Munson-Walker are not concordant and were affected by the amount of sugar solution taken. I n this instance the high figures were due t o a gummy residue which contained copper. The Munson-Walker method is superior in its definition of t h e time for bringing t o boiling, b u t Peters’ suggestion5 of a temperature limit is timely. The bulk of the reduction takes place before t h e mixture boils. On t h e whole, Bertrand’s method of reduction is preferred. As Bertrand’s tables did not reach below I O mg. sugar, t h e factor 0 . 4 9 was used t o convert copper percentages into sugar when necessary. R E M O V A L OF S I T K O G C X O I - s
norms
Bertrand’s method having been adopted as giving nearly accurate results for the purpose, various methods of removing t h e nitrogenous bodies which interfere were tested. Kearly all the work was done on meat extracts, which h a r e more of. the interfering bodies t h a n a n y other kind of meat product, and hence give the most severe tests of any method. Tt is well t o point out t h a t unclarified meat products give little or n o precipitation of copper because t h e cuprous oxide is held in solution b y t h e protein substances present. When these are partially removed a. point is reached a t which t h e holding of cuprous oxide in solution is reduced t o a minimum and high results are obtained, owing t o t h e presence of creatinin and similar substances which themselves cause a reduction of Fehling’s solution. While it cannot be said of a n y sample in advance which of these errors 1
8
4 5
“Estimation of Sugar in Blood,” J . B i d . Chem., 20 j1915), 67 G9. Chem. clbs., 10 (1916), 1230. .I. B i d . Chem., 24 (1916), 147. Jour. d s s o c . Oficial Agr. Chemists, 1, S o . 2 , .Aog , 1915 P P . 176 ~ 1 8 1 . J . A m . Chem. .SOL., 34 (19121. 930.
S o v . , 1916
T H E J O U R N A L O F IAVDiYSTRI.4L AiVD E , V G I N E E R I N G C H E M I S T R Y
will occur, i n general it is t h e case with meat extracts t h a t abnormally high precipitation of copper results. Hence with t h e methods here studied t h e lowest results are usually t h e most accurate. The details developed b y t h e writer for t h e use of either mercuric acetate or picric acid are as follows: METHOD I--MERCURIC
ACETATE
g'
free f r o m fat) with I j o cc. water I j or 20 min.; cool, a d d I O t o 2 0 cc. twice-normal mercuric acetate (slightly acid with acetic acid), t h e n a d d strong sodium hydroxide nearl:y to phenolphthalein, a n d make u p t o 2 0 0 cc., exclusive of fat. Filter through a large folded filter as quickly as possible. To 1 2 0 cc. of t h e filtrate a d d 2 cc. glacial acetic acid, a n d s a t u r a t e with H 2 S in the cold. Remove excess of H 2 S with a current of air, a n d restore volume t o 1 2 0 cc. Filter, t a k e 80 cc., a d d 5 cc. (or a n excess) of 20 per cent aqueous phosphotungstic acid a n d j cc. concentrated HC1, and make to Ioo cc, Filter and determine dextrose at once; 2 o c(;. of filtrate equal 3 , 2 o g. sample. Invert another portion of filtrate according t o Bureau of Chemistry Bull. 107,p. 41, revised, a n d deMEATS-Boi1
4O
termine total reducing sugars. T h e hydrochloric acid already added is not sufficient for complete inversion in t h e time stated. M E A T ExTRAcTs-~issolve g. extract in Ioo cc. water, add about 3 5 cc' mercuric acetate a n d proceed as with m.eats. T e n cc. or more of phosphotungstic acid solution should be used instead of five.1 ' Twenty cc. Of before inversion contains 400 mg. of sample. METHOD 2-PICRIC
ACID
31EATS-Boil jO g. finely divided sample (fairly free from f a t ) with about I j O cc. water f o r I j or 2 0 min.; cool, a d d I t o j g. picric acid (solid) a n d I j t o 2 0 cc. of 2 0 per cent aqueous solution of phosphotungstic acid, a n d make u p t o 2 j 0 C C . , exclusive of f a t . up to 160 cc' and take cc' Of with 8 cc. concentrated HC1 a n d 2 cc. water, mix, and filter. T h e filtrate in each case should .be clear. Determine reducing sugar a t once, 2 0 cc. of solution being assumed t o contain 3 . 7 5 g . sample. Invert a n d determine total reducing sugars. M E A T EXTRACTS-Dissolve 5 g. meat extract in about 2 5 cc. of water, a d d a n excess (4 t o 6 g.) solid picric acid a n d a n excess (40 t o 60 cc.) of a 2 0 per cent aqueous solution of phosphotungstic acid.2 N i x a n d ~ i l t t ~a k~e ,bo cc. (or more) make up to Ioo cc. cc. (or more, if necessary) concenof filtrate, a d d trated HC1, make up t o 66 cc. and filter quickly. The filtrate shoud be perfectly clear, b u t will have a light yellow color due t o picric acid in solution. Determine reducing sugar in t h e filtrate a t once. T w e n t y cc. equal 0.909 g. sample. T a k e another portion of t h e filtrate a n d invert, adding more HC1 according t o one of t h e methods in 1 An excess of hoth mercuric acetate a n d phosphotungstic acid is required. * An excess of both picric and phosphotungstic acids is required, b u t the hydrochloric acid should be kept as small in amount as the nature of t h e solution will permit.
IO25
Bureau of Chemistry Bull. 107, p. 41. Determine invert sugar. Results obtained b y these methods are given in Tables I a n d 11. Only a few representative figures are given, t h e others being entirely similar. The amounts of sugar recovered when known amounts of sucrose were added t o t h e sample before clarification indicate t h a t t h e methods as a whole are correct, t h e being no greatert h a n might be obtained on pure sugar solutions. It must be remembered t h a t very small amounts of sugar are being dealt with a n d that any is apt to appear relatively large. In one drop of thiosulfate solution was equivalent t o several hundredths of a per cent of sugar. When dextrose was added t o t h e filtrates after clarification, t h e errors inherent in t h e clarification were avoided. The figures show t h a t practically t h e same amount of sugar was recovered as was added, indicating t h a t , on t h e one hand, no cuprous oxide is held in solution, a n d on t h e other t h a t no nitrogenous or Other substance is present which causes increased reduction. TABLEI -PER Clarifier used
SAMPLE
CEKT
in Meat
SUGAR-RecovTotal ered Error
Added
SUGAR ADDEDt o ORIGINAL SAMPLE:
Sausage. , , . , , , , . , , , H g acetate 0 . 2 9 0 . 1 1 Sucrose Fresh M e a t Extract. H g acetate 1.77 Fresh Beef Extract ... Picric acid
4 . 9 Sucrose
0 . 6 5 1 0 . 0 Sucrose
SUGAR ADDEDro CLARIFIEDFiLrRArE: Diluted Beef Extract Hg acetate 0.58 7.44 Dextrose
Fresh M e a t Extract. Cured Meat Extract Cured M e a t Extract Fresh M e a t E x t r a c t ,
Hg Hg Hg Hg
acetate acetate acetate icetate
1.24 3.81 3.97 1 , 12
0.40
0.42 0.41 6.67 6.62 6.83 10.65 10.60 10.88 10.62 6.67
$0.02 $0.01 10.00 -0.05 +0.16 -0.05 $0.23 -0.03
8 . 0 2 7.95 -0.07 3.72Dextrose 4 . 9 6 4 . 9 9 $0.03 7.27 Dextrose 11 . 0 8 10.99 -0.09 3.72 Dextrose 7.69 7.69 1 0 . 0 0 0.96 Dextrose 2 . 0 8 2 . 0 6 -0.02
T h e preceding results having indicated t h a t sugar is quantitatively recovered b y these procedures. commercial samples of meats and meat extracts containing sugar were examined for reducing sugar before a n d after inversion, with t h e results shown i n Table 11: TABLE 11-COMPARISOB
BETWEEN MERCURYAND PICRIC ACID METHODS PERCENT SUGAR FOUND BEFOREINVERSJON AFTER INVERSION SAMPLE Hg Method Picric -Hg Method-Picric-
E z ~ ~ ~ , ~ f , ~ u r , e ,2d, 628, 3 26, 3 6
5,23 5 , 3 2 5,20
Ez';i,of,cur,e,d,,
6,84 7 , 0 j 6 , 8 j 7,10
,
,
Sugar cured h a m . . . . . . . . 0.11 Sugar cured bacon . . . . . . , 0 . 2 2 Extractof
,
,,,
,
, , ,
5.22
. . . _ 0.12
0.45
0.24
0.60
. . . _. . . . ( . . . . .. . ., , , .., ,
2,34
2,8j
,
, , , ,
$,?enss(,1~y~~'2,34 2 , 5 0
,
,
,
,
, ,,
,,, ,
6,96 ; , l o 0.46
....
0.60
,
.
.
2,90
These figures indicate t h a t t h e same results may be obtained b y both t h e mercuric acetate a n d t h e picric acid methods. -4s t h e latter is very much shorter, its superiority is Shown. In order t o make as severe a test as possible of t h e relative values of t h e various precipitants, I O O g. of extract which was known t o be chiefly from muscular tissue of fresh beef, a n d hence nearly free from sugar, were dissolved in water and several portions clarified as indicated in Table II1. At first the was not made complete, i n order t o test t h e necessity for t h e use of hydrochloric acid, and t o some extent, of phosphotungstic acid.
T H E J O U R N A L O F I N D U S T R I A L A N D EiVGIhTEERING C H E i t f l S T R Y
1026
1-Ilercuric acetate (already described) 2-Picric acid (already described) X E T H O D 3-Mercuric nitrate (otherwise same as I ) METHOD 4--A modification of Neuherg & Kerb’s rneth0d.l T h e solution was made alkaline t o phenolphthalein with strong solution of sodium carbonate and strong mercuric acetate added in excess. Considerable difficulty was experienced in ascertaining this point, more sodium carbonate being added once or twice. After filtering off t h e precipitate, t h e excess of mercury was removed a n d the solution treated as in Method I : METHOD
METHOD
TABLE 111-EFFICIENCY OB CLARIFYING AGENTS Per cent Phosphotungstic Test No. Sugar Method Acid 7 0.19 4 hTone I1 2.01 3 Xone 4 0.67 4 IL’one ~~. 0.63 4 Partial 1 0.69 2 Partial 12 I . 14 3 Partial
HC1 None (excess H g not removed) None x ”.n e ~
~~
&-one Partial Excess
COMPLETE C49RIFICATIOIi
13 2 5 3 9 10
0.74 0.45 0.60 0.50 0.58 0.56
__-
1 2 1
2 4 4
Excess Excess Excess Excess Excess Excess
Excess Excess Excess Excess Excess Excess
0 . 5 9 Average of completely clarified solutions 0 . 4 8 Average by Method 2
T h e results show the need of complete clarification, Method 3 giving especially high results. T h e other methods g ve comparable results, the error of t h e conditions worked under being probably more t h a n 0 . I per cent. Test S o . 7 was run without t h e removal of mercury, and was of no value. Methods 2 (picric acid) and 4 (sodium carbonate and mercuric acetate) gave the best results, t h e picric acid method being much t h e easiest a n d shortest of the four. From t h e experience of t h e writer, it is thought t h a t t h e average of Tests z and 3 was nearly t h e true amount of sugar in t h e extract ( 0 . 4 8 per cent). Dextrose was then added t o unused portions of solutions and t h e total reducing sugar determined. The results (Table IV) are perhaps more conclusive t h a n those in Table I I I ? as i t is easier t o estimate correctly t h e larger amounts of sugar. Three tests using no phosphotungstic or hydrochloric acid showed errors of 0 . 2 0 t o 0 . j 5 per cent. Four tests, using some phosphotungstic acid but no HC1, gave errors from 0 . I O t o 0 . jo per cent. Three tests (one b y each method except the mercuric nitrate), using excess of both phosphotungstic and hydrochloric acids, gave errors of only 0 . Q I t o 0 . I O per cent.
Vol. 8, KO.1 1
One important difference between mercury salts and picric acid should be noted. After recovering t h e mercury it is convenient t o add t h e phosphotungstic acid and t h e hydrochloric acid together. With picric acid, however, the reactions are somewhat different, three precipitates being formed: (I)-Picrates of protein substances, precipitated in solutions containing organic acids. (2)-Phosphotungstates insoluble in solutions containing no free mineral acids. (3)-Phosphotungstates soluble in neutral solutions or in t h e presence of organic acids, b u t insoluble in hydrochloric or sulfuric acid. T h e first two precipitates are not a,ntagonistic t o one another, b u t are more or less dissolved on t h e addition of hydrochloric acid. For this reason it is best t o add the picric and phosphotungstic acids together, filtering off t h e precipitate before the hydrochloric acid is added. Xitrogen determinations show t h a t about fivesixths of t h e total nitrogen is removed b y this process. As the nitrogen remaining is in compounds of low molecular weight such as ammonia and mon-amino acids, in reality nine-tenths or more of t h e nitrogenous substances are removed. Two extracts gave the following- results: Original nitrogen. .. . , , . . . 7 . 5 9 % Original nitrogen. . . . . . . , , 9.32% After picric acid., , . . . . . . 5 . 4 0 % After mercuric acetate, After phosphotungstic acid 2.18% phosphotungstic acid After hydrochloric acid ... . 1 . 2 8 % and H C I . , . , , . . . . . . . , , I . 2 1 % E R R O R I N R E D U C I N G S U G A R R E S U L T S D U E TO I N V E R S I O N
O F SUCROSE
T h e methods herein described are open t o t h e objection t h a t t h e hydrochloric acid inverts the cane sugar so rapidly as t o make the reducing sugar results too high. While this objection does not appear t o have good grounds, yet even if i t were true the methods would have value owing t o t h e facts t h a t the total sugar estimation is very accurate and t h a t t h e natural sugars in meat products are reducing. However, some careful tests of t h e amount of inversion have been made. It is well known t h a t even in neutral solutions sucrose reduces Fehling’s solution. T h e minimum amount of hydrochloric acid for complete precipitation of the phosphotungstates is about liqOththe total volume of t h e solution. When this amount is employed it appears t o be all used up in reactions with the other substances in t h e solution. Five cc. of one solution TABLE IV-RECOVERY O F ADDED DEXTROSE F R O M BEEF EXTRACT required 40 cc. of i V , / ~ o alkali for neutralization, P E R CENT SUGAR Sugar I n Extract Added Total Found Error Clarifier NO. b u t t h e volatile acid distilled from a similar portion (a) N o phosphotungstic o r hydrochloric acids required only 0 . 8 cc. 0.48 12.47 12.95 13.50 +0.55 h7azC03 + H g a c e t a t e 18 3.45 -0.20 H g nitrate 4- N a O H 0.48 3.17 3.65 23 Comparisons of t h e percentages of reducing sugar 5.10 -0.47 H g acetate -t N a O H 0.48 5.09 5,57 24 ( b ) Insufficient phosphotungstic acid: N o HCI obtained from meat extract solutions before and a f t e r 13.61 13.59 --0.50 Picricacid 0.48 14.09 15 0.48 3.36 3.84 3.65 -0.19 NalC03 + H g acetate t h e addition of cane sugar gave these results: 19 0 48 0.48
20 21
0.48 0.48 0.48
17 22 25
3,36 3.33 -0.49 N a L O s t H g acetate 3.84 3.36 3.84 3.94 +O.lO NazCO3 + H g a c e t a t e ( c ) Excess phosphotungstic acid and HC1 2.58 --0.01 Picricxid 2.11 2.59 4.06 4.48 4.54 -0.06 NaK03 i H g acetate 2.66 3.14 3.24 +O.lO IIg acetate 4- N a O H
As these figures are entirely in accord with all others obtained, they appear t o indicate t h a t these methods give accurate results, Sodium carbonate is better in conjunction with mercuric acetate t h a n sodium hydroxide, on account of the action of t h e Con. 1
Biochenz. 2 , 40 (19iZ!. 498-502.
Reducing sugar found in absence of sucrose .~~ Sample 1 1.64 Sample 2 0.49 Sample 3 1.54 Sample 4 0.20 ~
~~
~
Reducing sugar found in Dresence of sucrose after adding Sucrose 5 . 0 7 0 1.67 Sucrose 6 , 5 7 0 0.47 Sucrose 7 . 5 7 @ 1.49 Sucrose 1 0 , 0 % 0.20
In these cases t h e copper reduction was begun in 2 or 3 min. after t h e addition of t h e hydrochloric acid. T h e sugar was added t o t h e original product. When cane sugar has been added t o t h e solution after clarification some increase of reduction has been
T H E JOURNAL OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
Nov., 1916
observed, b u t not more t h a n would be obtained on aqueous solutions of sucrose. It is only necessary t o give one out of several series of tests. Meat ext r a c t solutions containing about 4 per cent of total sugar, mostly reducing, were each treated with 4 0 mg. sucrose (about j per cent) a n d allowed t o s t a n d a few minutes before neutralization. Reducing sugar was t h e n determined on this solution a n d also on a control solution identical except t h a t no sucrose was added. A blank test on 40 mg. cane sugar showed reduction equivalent t o 0 . 2 1 per cent sugar on t h e amount of sample used. T h e extract solutions gave t h e following results (percentages) : Time of standing Minutes 1
Sucrose present 5.27
4 2
4.94 5.00
RIDUCING SUGAR FOUXD I n (control I n sucrose solution Increase 3.95 4.16 0.21 4 . 2 28
4.41 4.35
3 4.92 4.30 4.46 Reduction of aqueous sucrose solution. . . . . . .
~:;~]Av.O.16
.. ,
0.16 0.21
T h e temperature of t h e room during this experiment was about 2 5 ' C., which was favorable t o inversion. T h e above figures show t h a t t h e estimation of reducing sugar in t h e presence of sucrose is quite practicable b y this method, t h e increased reduction being within t h e limits of error of ordinary sugar methods. It should be remembered also t h a t t h e solution is acid before t h e addition of hydrochloric acid, so t h a t although inversion is slow, in t h e presence of sucrose t h e whole process should be carried through without t o o much delay. After t h e addition of t h e hydrochloric acid t h e filtrate should be neutralized immediately. Where only total invert sugar is & a i r e d , t h e solutions do not change for several days. Clarification becomes more complete as t h e solution stands. It is believed t h a t t h e methods outlined i n this paper offer accurate means of determining reducing sugars i n meat extract before a n d after inversion. S U X MAR Y
I-Clarification of meat extract solutions for estimation of sugar b y Fehling's solution is best accomplished b y a n excess of picric a n d phosphotungstic acids, followed b y a minimum a m o u n t of hydrochloric acid. 11-In t h e presence of sucrose, reducing sugar may be determined within 0 . I or 0 . 2 per cent, provided proper precautions are observed. 111-Total reducing sugar may be determined within 0.1per cent.
u. s.
M E A T INSPECTIOK
LABORATORY
KANSASCITY, K A N ~ A S
LIGNOCERIC ACID FROM ROTTEN OAK WOOD By M. X. SULLIVAN Received July 3, 1916
I n t h e s t u d y of t h e origin of organic soil constituents a chemical s t u d y of rotten wood was undertaken in these laboratories in t h e year 1908. A large a m o u n t of dead oak wood was collected from a woody section near Washington, D. C. Since vanillin had long been reported1 as a constituent of wood, the ground-up wood was extracted with alcohol, t h e alcohol extract freed Singer, Monatsh , 3 (1882), 395.
1027
from alcohol a n d t h e residue treated with ether. F r o m t h e ether extract, b y t h e customary methods of testing for aldehydes, a small amount of a n aldehydecontaining resin of a vanillin odor was obtained which gave t h e color reactions of vanillin. Since vanillin is sublimable, about 2 0 0 g. of wood were heated between large watch-glasses t o obtain sublimate matter, a n d though vanillin was subsequently found in wood a n d other vegetable material b y appropriate methods,l practically all t h e material sublimed from t h e wood was insoluble in hot water and was of a f a t t y nature. T o obtain a large amount of this sublimable f a t t y matter, about 2 0 lbs. of rotten wood were placed in a retort, connected with a Liebig condenser, a n d subjected t o heat. As a result of regulated heating with Bunsen burners a steady stream of material varying from t a r r y matter t o a yellowish white crystalline solid distilled over through t h e condenser. T h e material obtained was soluble in hot alcohol. On cooling, crystalline matter separated out of t h e alcohol. On filtering while t h e alcohol was still warm a n d washing with cold alcohol, most of t h e coloring matter went into t h e filtrate. T h e crystalline matter was t h e n placed on a porous plate a n d treated with cold petroleum ether until colorless. T h e purified substance recrystallized from alcohol melted a t 737 5 ' . T h e compound was soluble in ether, hot alcohol, warm petroleum ether, a n d was insoluble i n water. Subsequently, b y much t h e same method, lignoceric acid2 was obtained from peat soil. Lignoceric acid has t h e elementary composition of C, 7 8 . 2 ; H, 13.0; 0, 8.8, As determined b y Dr. E. C. Shorey a t t h e time he was working on t h e lignoceric acid of peat soil, t h e elementary composition of t h e substance obtained b y me from wood was C, 7 8 . 6 ; H , 1 3 . 3 5 ; 0, 8 . 5 . I t was thought a t t h e time (1909) t h a t t h e substance from t h e wood might be identical with lignoceric acid which Hell3 found in beech wood t a r . T h e elementary composition a n d melting point, however, were suggestive t o me of cerotic acid from beeswax, which has a percentage composition of C, 78.78; H, 1 3 . 1 3 ; a n d melts a t 78'. No further work was done on t h e identification of t h e compound from t h e rotten wood until very recently, when t h e question of t h e identity of t h e wood substance was revived b y t h e finding of cerebrosides4 i n mold from soil a n d b y t h e work of Levene and West, who found t h a t lignoceric acid is a n oxidation product of cerebronic acid derived from cerebrosides of brains b y hydrolysis. Lignoceric acid forms a lithium salt insoluble in hot methyl alcohol. Accordingly, t h e material from t h e wood was dissolved i n methyl alcohol and the resulting solution was treated with a methyl alcohol solution of lithium acetate as long as a precipitate formed. I n this way t h e material collected from t h e Sullivan, THIS JOURNAL, 6 (1914), 919. 0. Schreiner and E. C. Shorey, U. S. Dept. Agr., Bureau of Soils, B U Z Z . 74 (1910). 3 Ber., 13 (1880), 1709. 4 Sullivan, Science, 38 (1913), 678. 6 J. Riol. Chem., 15 (1913), 193. 1
2
