Vol.
1720 [COXTRIBC?IOV
FROM THE
cis
CHEMICAL LABORATORY O F THEOHIO STATE UXIVERSITI']
Chromatographic Isolatbn of Cane Juice Constituents BY \IW. *. RINKLEY' .WD 11.L. WOLFROM The ,juice cspressible from the stalk of the sugar cane has long been known to contain reducing sugars,* whose amount decreases to maturity and then again increase^.^ Louisiana cane a t harvesting normally contains 1-1 .5y0 reducing sugars. .?tnalytical data and the concomitant presence of sucrose would indicate that these reducing sugars are probably D-glucose and D-fructose, but to our knowledge such sugars or their derivatives have never been isolated and adequately identified from this plant source. LVithout such an actual separation of a pure substance, or of a characteristic, crystalline and identifiable derivative, its presence in :t mixture cannot be considered as demonstrated. Prinsen Geerligs* separated from Javan cane juice a lime salt which on decomposition with carbon dioxide yielded a reducing sirup of large levorotation. He thus presented presumptive bu.t not definitive evidence for the presence of D-fructose. In the work herein described, chromatographic methotis4s5hare been applied to the problem of the identification of the reducing sugars present in cane juice. Kormal Louisiana cane juice solids were prepared by ice sublimation and acetylated. The acetate mixture was then chromatographed on a corn1ne:rcial hydrated inagnesiuin acid silicate a i n t l ii-glucose, D-fructose anti sucrose were identifictl as their crystalline acetates. In the course of this work i t was necessary to effect a chromatographic separation of the anomeric forms of D-glucopyranose pentaacetate. Control experiments indicated that the sucrose underwent no significant acetolysis under the acetylating conditions eii iployed. We believe that the general type of chromatographic procedure herein described is capable of wide extension to the sugar fraction of plant produci;s, th,e separation and identification of whose components have hitherto been difficult if not impossible.
Experimental Cane Juice.-~-.l'he unclarified or raw sugar cane (S(lc.c#$cina~.uwiL., variety Coimbatore 290) juice was collected a n d expressed in Louisiana in Sovember, 1945. I t was quick-frozen, packed in solid carbon dioxide a n d shipped t o Columbus, Ohio, where i t was still frozen on arrival. ciznrziiiz
.4naZ. (?& original juice) solids, 12.2; ash, 0.3; sucrose, 0 . 4 ; reducing sugars (as invert sugar), 1 . 1 ; PH, 6.0; d , 1.017 g. per ml. a t 25 '. Acetylation of Cane Juice Solids.-The cane juice solids were prepared b y desiccation through ice sublimation ("lyophilization"). An amount of 5.8 g. of cane juice solids was added slowly, with mechanical stirring, at - 10 t o a n acetylating mixture of 30 cc. of acetic anhydride and 0.5 g. of freshly fused zinc chloride. T h e temperature of the reaction mixture was maintained at -15 t o -10" for three hours and was then allowed t o rise t o 1 5 " over a period of eighteen hours. T h e unreacted solids (3.2 g.) were removed b y decantation a n d t h e acetylation mixture was poured slowly into 400 g. of ice and water. This solution was adjusted to a pH of 6.3 with sodium bicarbonate and n-as then extracted with chloroform. T h e extract was dried and t h e chloroform removed below room temperature. A viscous, golden sirup resulted; yield 4.0 g. A water-ethanol solution of the unreacted solids gave crystals which melted a t 180-182" (unchanged on admixture with an authentic specimen of sucrose of like melting p o i n t ) , [ O I ] ~ ~+66.9", D (c 2.7, water). T h e crystals are thus identified as sucrose. First Chromatography of Acetylated Cane Juice Solids.An amount of 2 . 0 g. of the above-described acetylated cane juice solids was dissolved in 2 5 cc. of benzene and t h e solution added a t the top of a 235 X 52 m m . column6 of a mixture (170 g.) of 5 parts (by weight) of "Magnesol"7 and 1 part of "Celite "8 T h e chromatogram was developed with l2,50 cc. of 100/1 benzeneg/ethanol10 (volume ratio), t h e column extruded and streaked with a freshly prepared aqueous solution of 1% potassium permanganate in 2.5 S sodium hydroxide. Six distinct zones were formed and these were c u t out (the indicator strcak was removed with a scalpel) and eluted with acetone. Pertinent d a t a relative to thc material in thew zones are listed in Table I . TABLE I FIRSTCHROMATOGRAM O F ACETYLATED CANE JUICE Approx. time, sec., for distinct Zone zone position in forma- mm. from tion in top of column indicator (232 mm. Zone streak long)
0-4 16-19
g.
Nature of zone material Amorphous solid Amorphous solid Partially ~ r y s t . ~ Cryst. 8-D-fructose tetraacetate C&yst. sucrose octaacetate Cryst. n-o-glucopyranose pentaacetate; 8-0-glncopyranose pentaacetate
D C
42
--
34-38 49-53
0,042 .053 ,019 ,085
B
60 "0
90-139 161-163
0.256
F E
I
x
I)
1,468
-
Total, 1 . 9 2 3 ( 9 6 . 2 C ) . 0
(1) Sugar Research Foundation Research Associate of T h e Ohio S t a t e University Research Foundation. (2) H. W. Wiley, B d . Chem. SOC.TYashinglon, 4, 2 2 (1890); Chem. Z e n i r . , 61, I, 354 (1890); H. C. Prinsen Geerligs, C h e m . - Z e d . , 20, 721 (1896). ( 3 ) F. A. F. C. Went (and H. C. Prinsen Geerligs), Jahrb. wiss. Botan., 31, 289 (1898); H. W. Wiley, THISJ O U R X A L , 26, 855 (1903). (4) W. H. McNeely, W. W. Binkley and M.'L. Wolfrom, ibid., 67, 527 (1945). (j) W. W. Binkley, M a r y G. Blair and M. L. Wolfrom, i b i d . , 67, 1789 (194:)).
120 240
Zone yield,
SOLlDS
A11;ount of crystalline material too small for itletiti-
fication. ( 6 ) Column dimensions refer t o t h e adsorbent. ( 7 ) A synthetic, hydrated magnesium acid silicate manufactured by t h e LVestvaco Chlorine Products Co., South Charleston, West Virginia. Only material passing a 200-mesh screen was employed. (8) No. 5 3 5 , a siliceous filter-aid manufactured by Johns-Manville Co., New York, N. Y. (9) 411 benzene employed mas free of thiophene. (10) Absolute ethanol w a s employed in all chromatographic operations.
Sept., 194-G
STABILITY O F
METHOXYL GROUPSI N METHYLATED HCl
glucopyranose pentaacetate and 0-D-Glucopyranose Pentaacetai:e from Zone A,-A solution of 80 mg. of t h e material from zone -4 in 2 cc. of benzene was placed on a 1iO X 35 mrn. column6 of 5:1 Magnesol/Celite and developed with 650 cc. of 250,'l benzene/ethanol. Two distinct zones were obtained a t a distance of 116-130 mm. and 1 B R - - l n 3 m m . from t h e top of t h e adsorbent column. T h e two zoiies wert cut out and eluted with acetone; yields 27 m g . (uppcr zoiiel aud 51 mg. (lower zone). T h e above experiment was repeated several times in order t o ohtain further material. After several recrystallizations from ethanol (9Sc;i the material from t h e lower zone was itlen-:ified as 3-~-glucopyranosepentaacetate; m . p. 132" (mixed in. p. uncharigedi, [ ( Y ] * ~ D+do (c 3.9, red valucsll: m . p. 132", [ ~ ] ; O D +4". recrystallized material from t h e upper 1 a i wo-glucopyranose pentaacetate; in. p. 112- 113' (iniscd in. p. unchanged), [ a ] : * + ~l o l o (c 4.:3, clilorofoiin I ; acccptcd values": ni. p , 1 1 3 O , [ ~ ] ' O D +lOl.ii'. Sucrose Octaacetate from Zone B.-The material (1.47 g.) from zone El was recrystallized from 10 cc. of ethanol ( 9 5 7 ) ; yield 1.26 g., m.p. 5'5' unchanged on admixture with an authciitic ipecimen of sucrose octaacetate of like in. p . , [ a ] ? * $00' ~) i c 4.1, chloroforni). These d a t a identify the iulxtance as the higher-melting dimorph of sucrose octaacetate for which the accepted constants are: m . p . Sgo," [ a I r o+fiO" ~ (chlorofo~m).~~ $-D-Fructopyranose Tetraacetate from Zone C.-The material iO.083 9.1 from zone C was rexystallizcd from 3 (11) C. S. Hudson and J . K . Dale, THISJ O U R N A L , 37, 1261 (1915). (12) R 1'. I.iIi:,tead, A. Kutenberg, \V. G . Dauben and R'. L. Evans, ;bid. 62, 3260 19411, if X I . FrSrejscque, C o m p l . rewd., 203, 731 (193G' (13) C S H u d s o n and J. 31. Johnson, THISJ O U R K A L , 37, 2718 (I!) 15).
SPRUCE
LIGNIN
1731
cc. of ethanol (95%) ; yield 27 mg. of prismatic crystals, m . p . 130" (mixed m . p. unchanged), [ m I z 5 ~ -91.2" (c 3.9,chloroform). These d a t a identify t h e substance as 6-D-fructopyranose tetraacetate for which the accepted constants are: m . p. 131-132', [CY]"D -91.6" (chloroform). Control Experiment with P u r e Sucrose.-The acetylation and chromatographic procedure described above for the cane juice solids mas applied t o a sample of pure sucrosei4 (3 g.) There was obtained 4 mg. of crystalline material in zone C (fl-D-fruCtOpyrallOSetetraacetate zone) and 5 mg. in zone -4(D-glUCOpyranOSe pentaacetate zone)
Acknowledgment.-Acknowledgment is made to the laboratory assistance of hlr. John M. Kolbas and hliss Eloise L. Carpenter. The cane juice was kindly supplied by Dr. A. G. Keller of Louisiana State University, Baton Rouge. We are indebted to the counsel of Dr. R. C. Hockett, Scientific Director of the Sugar Research Foundation. Summary D-Glucose (as a-D-glucopyranose pentaacetate and P-D-glucopyranose pentaacetate), D-fructose (as P-D-fructopyranose tetraacetate) and sucrose (as sucrose octaacetate) have been isolated from sugar cane juice by chromatographic methods. (14) T h e sucrose was obtained from t h e Coleman and Bell Co , iXorwood, Ohio; i t s maximum invert sugar content was stated by t h e manufacturers t o be 0 23570.
COLUMBUS, OHIO
RECEIVED R.IARCH 13, 1936
[COSTRIBUTIOS FROM THEIXSTITUTE OF PAPER CHEMISTRY ]
The Stability of the Methoxyl Groups in Methylated Hydrochloric Acid Spruce Lignin1 BY F. E. BRAUNS In an earlier investigation on the decomposition of fully methylated spruce wood,la i t was found that lignin derivatives were obtained with a considerably lower methoxyl content than was expected. This might be caused by any one of the following possibilities : (1) the lignin was not completely methylated; (2) the lignin is chemically combined wi?h another component of the wood through an oxygen linkage, thus covering the hydroxyl group and preventing i t from being methylated; or ( 3 ) methoxyl groups are split off from the methylated lignin during its isolation. T o study this question, the behavior of fully methylated hydrochloric acid lignin toward a number of reagents used for the isolation of lignin was investiga.ted. The methylated spruce lignin used was obtained by methylation of hydrochloric acid spruce lignin which was prepared according to Kalb and Lieser2 \ 1) Presented before t h e Division of Cellulose Chemistry a t t h e lUVth meeting of t h e American Chemical Society, Atlantic City, S e w Jerbey. April 8 t o 12, 1046. , l a ) F E: Brauns and J , J . Yirak, in press. i % ) 1, Kalb iinc 1'.Lieser. B e y . , 61, 1007 (1928).
and was kept in a moist condition. After five methylations, a methoxyl content of 32.26% was obtained, corresponding to 9.8 methoxyl groups per lignin building unit (Table I). To obtain lignin derivatives soluble in organic solvents for further purification, the methylated hydrochloric acid lignin was treated with reagents under conditions previously used with unmethylated hydrochloric acid lignin and also in the isolation of lignin from wood. Extracted spruce wood and hydrochloric acid spruce lignin, when treated with acetic acid in the presence of a small amount of magnesium chloride, give acetic acid spruce lignin.3 This lignin still contains all of its original methoxyl groups, but two of its hydroxyl groups are acetylated. Because the acetic acid hydrochloric acid lignin is soluble in dilute sodium hydroxide, the phenolic hydroxyl group must still be free. Upon methylation with diazomethane, the acetic acid hydrochloric acid lignin becomes insoluble in dilute (31 F E Brauns and hl A Buchanan, TIUS J O U R \ A L ( 19 4 3 1
67, 645
