170
ANALYTICAL CHEMISTRY Browning, P. E., and Plamer, H. E., 2. anorg. Chem., 59, 71 11908). Ibid., 62, 218 (1909). Charlot, G., Bull. SOC. chim., 6, 977, 1447 (1939). . . CibaRev., 4, 1391 (1941). Conant, J. B., Aston, J. G., and Tongberg, C. O., J . Am. Chem. Soc., 52, 407 (1930). Ekker, M. E. H., Rec. trav.chim., 13,36 (1894). Endis, D. T., and Becker, H. C., IND.ENG.CHEM.,AN.AL.ED., 15,262 (1943). Fenton, H. J. H., J . Chem. Soc., 1878,T 300. Ibid., 1879,T 12. Ibid., 1878,T 470. Foster, W., Ihid., 1879,T 119. Fresno, C. del, and Mairlot, E., Anales SOC. espaR. jis. ywim., 30, 254 (1932). Ibzd., 31,122 (1933). Ibid., 31, 531 (1933). Ihid., 32,280 (1934). Fresno, C. del, and Mairlot, E., Rev. acad. cienc., Madrid, 30, 315 (1933). Fresno, C. del, and Valdes, L., 2. anorg. Chem.. 183, 251, 258 (1929). Gentele, J. G., Dinglers polytech. J., 152, 68 (1859). Ionescu, 9., and Vargolici, V.,Bull. soc. chim., Romania, 2, 38 (1920). Lenssen, E., J . prakt. Chem., 80, 408 (1860). Palmer, H. E., 2. anorg. Chem., 67, 317 (1910). Ramsey, 3. B., and Robinson, A., J . Am. Chem. Soc., 52, 480 (1930). Scapliarini, G., S t t i X congr. intern. chim., 3, 466 (1939). Thielke, R. C., dissertation, University of Michigan, 1935. TomicPlr, O., Arhiv Hem. Technol., 12,105 (1938); Chem. Listy, 32, 442 (1938). TomicPk. O., Rec. trav. chim., 44, 410 (1925). TomicPk, O., and Freiberger, F., J. Am. Chem. Soc., 57, 801 (1935). Willard, H.H., and Cake, W. E., Ibid., 43, 1610 (1921).
ferricyanide and vanadyl sulfate. Some indicators which were satisfactory. with the other substances analyzed failed t o give a detectable end point. The results given in the table are the corrected values. A comparison of the diphenylamine derivatives studied is givenin the fourth column of Tables I1 and 111. The usefulness of each compound was det'ermined by taking into consideration precision, accuracy, sharpness of the end point, and feasibility, and the number of plus signs indicates the increasing desirabilit,y of each indicator in the titrations. The minus sign means that the indicator was tried but the results were not worth considering. I n most cases the end point recorded by the indicator in t'hese esperiments is for the reaction between ferricyanide and vanadyl sulfate. Slight deviations from the standards may be due to differences in the readiness with which ferricyanide oxidizes the different substances. Although 2,2'-dicaiboxydiphenylamine exhibits a vivid, sharp end point, except with chromium, it did not give reasonable precision. The halogen-substituted carboxydiphenylamines were not so useful as the methoxy and methyl carboxydiphenylanlincs. Introduction of the amino group in the diphenylamine sulfonic acid molecule does not improve the indicator for the ferricyanidevanadyl sulfate titration. However, with the hypobromite-arsenite system aininodiphenylamine sulfonic acid is slightly better t'han diphenylamine sulfonic acid. The indicator blank of the former derivative is much smaller with hypobromite than with ferricyanide. The reverse is true for diphenylamine sulfonic acid indicator.
\_._.,.
LITERATURE CITED
(1) Becker, H. C., and Englis, D. T., IND.ENG.CHEM..AS.AL. ED.,
13,15 (1941). (2) Bollenbach, H., and Luchmann, E., 2. anorg. Chem., 60, 446 (1909). (3) Britton, H. T. S., and Konigstein, H., J. Chem. Soc., 1940, 673.
FROM a thesis presented by Gloria D. AManaloto the Graduate School of the University of Michigan in partial fulfillment of the requirements for the degree of doctor of'philosophy.
Determination of Carotene in Alfalfa Studies on Methods f o r Meals and Fresh Leaves F. P. ZSCHEILE'AND R. A. WHITMORE,Department of Botany, University of Chicago, Chicago, I l l .
.
,
Methods are presented for the rapid, simple, and precise extraction and determination of carotene in fresh alfalfa leaves and in dried alfalfa meals. Numerous tests were made to clarify details of procedure. The carotene fractions of fresh and dried alfalfa were studied chromatographically and spectroscopically. Neo-B-carotene B was found in fresh alfalfa in addition to all-trans &carotene. In dehydrated alfalfa members of the neo-&carotene U, V, W group are also present. Blanched leaves may be refrigerated for future analysis after collecting trips. The method is precise to 3 0 . 5 to 1%.
A
RAPID and simple method for routine determination of the carotene fraction in fresh alfalfa and in alfalfa products was required in this laboratory. I n this study the advantages and disadvantages of many detailed parts of different procedures have been considered carefully. The methods described below have proved satisfactory and a critical discussion of various points may be helpful, since the analysis of alfalfa products is recognized as difficult. KO attempt is made here t o review the literature extensively or t o discuss all existing methods. I n recent years numerous modifications have been described which are different combinations of well-known principles. The carotene fraction contains the pigments of alfalfa which may possess vitamin A potency, in contrast to the xanthophylls, oxidation products of carotenoids, chlorophyll, and its decompo1 Present address, College of Agriculture, University of California, Davis, Calif.
sition products. For all practical purposes, the carotene fraction of alfalfa may be considered primarily a mixture of all-trans p-carotene and its cis-isomers. EXTRACTION
Fresh Alfalfa Leaves.
Five grams of fresh hand-picked leaves are wrapped in a cheesecloth package, tied with string or wire, and blanched by immersion in boiling water for 5 t o 10 minutes. Excess water is removed by application of moderate pressure. Analysis may follow soon, or the sample may be frozen with dry ice for future analysis. Carotene is extracted by blending 5 minutes in a Waring Blendor with 100 ml. of 40% (by volume) acetone in Skellysolve B in the presence of approximately 0.1 gram of magnesium carbonate. After 3 minutes of blending, the blender is stopped and the sides are cleaned by wiping downward with a piece of filter paper, which is then shredded and added t o the mixture. Blending is continued 2 minutes more.
V O L U M E 19, NO. 3, M A R C H 1 9 4 7 The mixture is filtered under suction and the residue is washed ively Ivith two 10-ml. portions of acetone, followed by olve B until the filtrate is colorless. The filtrate is transferred to a separatory funnel, the aqueous layer removed, and the hyperphase made t o 200-ml. volume with Skellysolve B. S o drying is necessary. A 15-ml. aliquot of this solution is diluted with 15 nil. cif Skellysolve B, and 10 t o 20 ml. of the resultant solution are chromatographed as described below. Dehydrated or Dried Alfalfa Meal. TKOgrams of dry meal (ground t o 40-mesh) are used. T h e sample is placed between layers of cotton in a Coors porcelain extraction crucible, size 3, suspended in an ,I.?.T.M. extraction apparatus. Sixty milliliters of 30% acetone-Skellysolve B are put in the 400-ml. flask of the extraction apparatus and the sample is extracted hot for 3 hours on a steam-heated bath (with glycerol, or mineral oil for rapid heat transfer), with a drip rate of 100 t o 150 drops per minute. This is a modification of the method of Wall and Kelley (6). The extract is made to 100 ml. with Skellysolve B, resulting i n a final acetone concentration of about 18%; no filtering or drying is required, althoush the solution is usually turbid with precipitated material. An aliquot of this solution, usually 15 ml. (from 5 t o 20 ml. may be used), i5 chromatographed as described below. CHROiMATOGRAPHY
.4 short (about 6-em.) column, 19 mm. in diameter, composed of a 1 t o 1 mixture of activated magnesia (Micron brand KO. 2642, Westvaco Chlorine Products Company, Sewark, Calif.) and Hyflo Super-cel is packed dry with a wooden tamper, and then covered with 1 cm. of anhydrous granular sodium sulfate. Under these conditions the carotenes pass rapidly through the column and the chlorophylls, xanthophylls, and carotenoid oxidation products are held firmly in the upper 10 mm. of the column. The eluate from the column is cauTht in a 25-ml, volumetric flask, using a Fisher Filtrator, and the column is washed with 10% acetone-Skellysolve B to make the volume of the eluate to almost 25 ml. T h e upper zones expand to a depth of about 2 em. during this washing. Table I.
Effect of Cold Storage (below- -18' C.) on Carotene Content of Fresh Alfalfa Leaves Carotene Content (Fresh Weight Basis)
Storage Time Dam
P.0.m 163 162 166 164
1
21
30 66
PHOTOMETRY
The density of the 25-ml. solution is determined a t 436 nip n i t h a Coleman Universal spectrophotometer, Model 11, or a Iilett photometer with a S o . 44 filter, and converted into carotene concentration by a calibration chart. Density values are between 0.15 and 0.95 and between 0.08 and 0.60 for the Coleman and Klett photometers, respectively, for samples having bet?$een 50 and 400 p.p.m. of carotene (15 ml. chromatographed). These instruments were calibrated by use of a carotene fraction (from alfalfa meal chromatographed on magnesia) which had a characteristic absorption curve like that reported by Beadle and Zscheile (1) for the carotene fraction of spinach leaves. Thia curve is intermediate between thoqe for all-trans &carotene and neo-&carotene B (1, 7 ) . The concentration of caqtene is measured with a Beckman spectrophotometer at 4360 A. using t h e specific absorption coefficient 196 ( 1 ) . DISCUSSION OF CRITICAL FACTORS
Different plants, plant parts, or products made from plants require individual treatment and study, as extraction method
