818
ANALYTICAL CHEMISTRY
parallel that of aconitic. Somc of them limy pi acticall) disappear when the plant ripens, although this assumption requires vel ification. It is difficult to explain Tanabe’s (IS) failure to isolate succinic or fumaric acids, which were specifically sought in his examination of juice which yielded appreciable quantities of aconitic acid. 308
I
approximately the same in tliffei,ent varieties. The agreement in composition of this organic acid frwtion of the plant cane and the stubble, or second growth cane of variety Canal Point 44-154 is striking. It is more remarkable in view of the fact that the second growth cane was harvested almost a month earlier than the sample of first year plant cane used. This result may be a coincidence and will require checking hy more extensive analyses of plant and stubble cane juicw. Aconitic acid determinations were made directly on the juice samples in the course of other work on the processing of these sugar cane varieties, rather thaii on the same lyophilized juiw solids prepared for chromatographic. analysis. Agreement of the values obt,ained by the decarbosylation method Kith those determined chromat’ographically are good except in the case of variety Canal Point 44-101, in which aconitic acid and all of the other acids are present in the smallest amounts. I n this case the chromatographic value is probably more accurate, as the decarboxylation method is subject to errors of constant magnitude i n the deterniination of small amounts of carbon dioxide and is less reliable than chromatography for analysis of juice samples cont:tining lrss than 1% aconitic acid nn solids. ACKNOWLE1)GMENT
2000
4000
6000
8000
10000
MILLILITERS O F EFFLUENT
Figure 2.
Effect of Chloroform Extraction on EtherExtractable Acids
Data pertaining to the separation of the acids foi, qualitative identification are assembled in Table I, which sho\vs t,he peak effluent volumes as defined by AIarvel and Rands for the frartionP obtained by chromatography of various concentrated preparations of juice solids. The results show that some of the acids may be lost by certain procedures used for preliminary conrentrations : neverthelcss, concentration is necessary so that rharges of practical amounts of less abundant acids may be applied to tho columns without overloading them with the very large e s w s s of aconitic acid present in these juice samples. All nine acids dtBt,ectable by the direct analyticul technique were collected 11y Permutit-S anion exchanger :ind could be separated from t,he regenerate by applying the totul aridified solids mixture dirertly to the chromatographic c:olunin. \Vheri the organic acids x e r o extracted from the acidified regenerate solution with ether, larger charges could be used but citric acid was lost in this step because of its insolubility in ether. Tlir further chloroform extraction of the ether-extractable acids necessary to eliminate the bulk of the aconitic acid resulted in the loss of glycolic and o d i c ac%l!: in addit,ion to the citric acid, as shown in Figure 2. The aqueous solution remaining after ether extraction readily yiplded ritric acid upon concentrat,ion, extract,ion of the sirupy concentrate with acetone, and chromatographic purification. Suffirient quantities were thus obtained for crystallization and preparation of derivatives. Because of their similarity in solubility properties to aconitic acid, the accumulation of glycolic and oxalic acids presented greater difficulty, but both of these have been independently detected previously by other methods. The excellent agreement of the peak effluent volume found for glycolic acid in this work with that determined by lIarvel and Rands provides satisfactory characterhation of this acid, supported by the paper chroni:~tographicidentification of Wiggins. The properties of oxalic acid p l a c ~it at the limit of dt‘ectivcness of the partition method of itleiitification but the pi’esence of this acid in sugar cane has been fully estnblished by scveral workers using standard analytical procedures (10, 13, I ? ) . The results of analyses of individual varieties of cane aesenihted in Table 111 are preliminary and itre given to indicate the applicability of the method to projected investigations of the composition of sugar cane juice. These limited data show that’ the vxieties differ in the total concentrations of the acids determinrd but that the relative pi,o~iortionsof individual :irids are
The authors are indebted to C. .A. Fort,, B. A. Smith, and I$-,I?. Guilbeau, of the Sugarcane Products DiviBion, for their assistance in preparing the ion exchanger regenerates and lyophilized juices, and to Lawrence E. Brown, of the Analyt~ic.al.Phy,qical Chemical, and Physical Division, for microandyses. LITER i r w E CITED
dmbler, J. A., and Roberts. E . ,l,,. k < r . . CHRX, 19, 877 (1947). Behr, Arno, Ber., 10, 351 (1877). Bulen, W.A., Varner, J. E., an(l lhirrell. 11. C.,.4xu3.CHEM., 2 4 , 1 8 7 (1952).
Claborn, H. V., and Pattewon, W , I., .I. ASROC.Offir. A y r . Chemists, 31, 134 (1948). Fort, C. .4.,Smith, B. -4.,13l:ic.k. (’. I. .. and Xhrtiti 1,. F., Sugar, 47, h-0. 1 0 , 3 3 (1952).
Guilbeau, W. F., Black, C. L., atid AIartin, I,. I?., Sicgar J . , 14. S o . 6. 18 (1951).
Rei&, A. G., and Schafer, F. C . , Loriisinn!i State Cniv., I h g . Expt. Sta., Bull. 24 (1951). Xlarvel, C. S.,and Rands, 11. I),, ,TI., .I. .4m, f ’ / i v w , .Sot... 72, 2642 (1950).
Seish, A. C., Can. .I. R e s e a d t , 27B, 6 ( 1 949). Payen, -4., Compt. rend., 28, 013 (1849). Shorey, E. C., J . Am. Chem. Soc.. 2 1 . 4 5 (1899). Takei, S., and Imaki. T.. H u l l . 1mt. Phus. (”hvm. Ruscawh (Tokvo), 15, 1055 (1936). Tanabe, T., Rept. Il’uirc’nrr Sicyui. E.rpt. Stu. (Formosa). So, 4, 33 (1937).
Wiggins, L. F., Intoii. Sirgar I . , 54,324 (1952). Winter, H., 2. V e r . deict. Z i k h r - I r d , 38, 780 (1888). Wise, W. S., Anal!la/.76, 816 (1951). Yoder, P. A , , I d , Ettu. ( ‘ h p n i . , 3 , 640 (1911). R E C E I V Efor D review October 5 , 103X. . h r e p t e d February 10, 1954. h e sented as part of the Yyiiijiosiiilii on Analytical .\lethods before the Di\-isions of hnalytical Chemistry and Carbohydrate Chemistry at the 124th XIeetinr of the .i\rtxxcas CHi;\rrcar. SOCIETT, (’liicago, I l l . , 19.53. Hefrrences to specific 1)rodrirtri of colir~iir’icialinanufarture are for illustration only and do not constitute endorsrrrirnt by the U . 9. Department of Agririittuw of the 1)rodIlcts na~rird.
Automatic Spectrophotometric Titrations-Correction In the article on automatic^ Spc,ctropliotonietri(: Titr;itinns” [llalnistadt, H. V., and Gohrbandt, I
