128
INDUSTRIAL AND ENGINEERING CHEMISTRY
Table IV gives the concentration of extraneous materials in the extract which may cause errors of 5 and of 10 per cent in the measured starch concentration, assuming a cornstarch concentration of 0.1 gram per liter. The data are derived from Tables I1 and 111. Columns 2 and 3 are calculated on the assumption that cornstarch a t a concentration of 0.1 gam per liter is and are on the assumption that a 5-gram Of paper is used, that the cornstarch content is 2 Der cent, and that the extract is diluted to 1 liter. Extraneous materials which must be present in quantities less than to per cent Of the paper IV)may serious error in the starch determination. Normally these materials are present to an extent of less than 1 per cent, but this is known to be true’ the accuracy Of the starch determination within the indicated limits cannot be assumed. Considerable error may be expected if the paper
VOL. 11, NO. 3
contains acid-soluble fillers and the starch is removed by acid extraction. (I) (2)
Literature Cited Euler, H., and Bergman, s., Ko&&%Z., 31, 81-9 (1922). Hanausek, T. F.,“Microscopy of Technical Products,” New
York, John Wiley & Sons, 1907. (3) Hardy, A. C., J. Optical sot. Am., 18, 96-117 (1929). (4) Miiller, R. H., and McKenna, M. K., J. Am. Chem. Soo., 58, 1017-20 (1936). ( 5 ) Paloheimo,’L., and Antila, I., Biochem. Z., 238, 401-7 (1931). (6) Pucher, G . and Vickew, H. B.3 IXD. ENG.CHEM.,Anal. Ed., 8, 92-7 (1936). (7) Reichert, E. T., Caarnegie Inst. Wash. Pub.173 (1913). (8) Sjostrom, 0. A,. IND. ENQ.CHEM.,28, 63-74 (1936). (9) Taylor, T. C., and Iddles, H. A., Ibid., 18, 713-7 (1926).
w.,
R E C E I V ~September D 21, 1938. Presented before the Division of Physical and ~~~~~~~i~Chemistry at the 96th Meeting of the Amerioan Chemical Society, Milwaukee, Wis., September 6 t o 9, 1938.
Carotenoids in Yellow Corn LORAN 0. BUXTON, National Oil Products Co., Harrison, N. J.
I
T IS WELL known that the vitamin A activity of yellow corn is due to the presence of carotene and cryptoxanthin, and not to the presence of the free alcohol or ester forms of vitamin A. As early as 1919, Steenbock (6) demonstrated that yellow corn was a better source of provitamin A than white corn. The experiments of Coward (3) in 1923 showed that large quantities of yellow corn are required to produce growth in rats which have been depleted of the fat-soluble vitamin A. Karrer and eo-workers (4) isolated zeaxanthin, a xanthophyll, which was found to be devoid of vitamin A activity when fed to rats. Kuhn and Grundmann (6) associated the vitamin A potency of yellow corn chiefly with cryptoxanthin. The present investigation was undertaken primarily to develop a suitable quantitative method for determining the provitamin A content of commercial samples of yellow corn. Various biological as well as colorimetric and spectrophotometric methods have been described by numerous investigators; however, none has proved entirely satisfactory as a simple, rapid, and accurate method giving reproducible results. One of the latest proposed methods is that of Clark and Gring (a), who published data on the spectrophotometric estimation of carotenoids in yellow corn. Their samples were prepared for carotene-cryptoxanthin analysis by extraction of the pigments from the ground corn with methanol, followed by saponification. The carotene and cryptoxanthin were then separated from the xanthophylls by distribution between low-boiling petroleum ether and 90 per cent methanol. The concentration of carotene and cryptoxanthin in the epiphase was detezmined by means of optical density measurements a t 4500 A. Spectrophotometric Apparatus The modified Bausch and Lomb visual spectrophotometer used by Buxton and Dombrow (1) was employed for determining the carotene-cryptoxanthin content of the unsaponifiable fraction of yellow corn samples. The concentration of carotene and cryptoxanthin was determined from the intensities of the absorption band a t 4500 A. by taking the mean of several readings. The kxtinction coefficient, E: Fm., at 4500 A. for pure @-carotene in heptane is 2380 (1). A
typical absorption curve (Figure I) for the nonsaponifiable portion of yellow corn, determined in heptane on a sample of Reid’s Yellow Dent, was photographed with a Bausch & Lomb medium-sized quartz spectrophotometer equipped with a Hilger rotating sector disk and a quartz biprism. The light source was a hydrogen-discharge tube. The wave length 4500 A. was found most desirable for determining the extinction coefficients on the carotene and cryptoxanthin fractions. Since the extinction coefficients for p-carotene and/or cryptoxanthin a t 4500 8. in heptane are essentially the same (Figure l ) , i t is possible to use the following formula for calculating the carotene and/or cryptoxanthin for a l per cent solution: (S X FIR X C ) = gamma of carotene and/or cryptoxanthin for a 1 per cent solution
where
S = the screen factor
F = the extinction coefficient for pure p-carotene or cryp-
toxanthin in heptane R = the reading expressed in centimeters C = the concentration
Experimental Procedure Weigh accurately into a digestion flask 20 grams of finely ground yellow corn and add 200 ml. of 5 per cent methanolic potassium hydroxide. Reflux on a hot plate or steam bath for at least one hour. Agitate occasionallyt o facilitate thorough digestion. Cool, allow the sediment t o settle, and decant the supernatant liquid into a separatory funnel containing 50 ml. of water. Extract the residual sediment until the washings are colorless (usually five or six extractions are sufficient) with 50-ml. portions of purified technical heptane (1). Combine the heptane and alcoholic fractions and shake thoroughly. Remove the alcoholic layer and re-extract with 50 ml. of heptane. Combine the heptane extracts and wash free from xanthophylls and alkali by shaking thoroughly with 100-ml. portions of 90 per cent methanol; reextract the first 90 per cent methanol wash with 50 ml. of heptane. To ensure thorough washing, examine the last methanol wash for free alkali by testing a few milliliters with phenol hthalein. Distill the heptane portion to a small volume un&r reduced pressure in the presence of nitrogen gas. Add a few milliliters of is0 ropanol to the heptane solution before distillation t o facilitate t i e removal of water. Make up the concentrated carotenecyptoxanthin solution t o volume (50 ml.) with heptane and determine the intensity of absorption at 4500 A. with the visual spectrophotometer.
’
MARCH 15, 1939
ANALYTICAL EDITION
129
fraction, it was possible to calculate by difference the concentration of -1.6 cryptoxanthin present. No attempt was made in this investigation to elute the cryptoxanthin from the adsorbent other than to prove that i t was homogeneous and free from carotene. -1.2 All the samples used for these in".(' vestigations were obtained from the Nopco Experiment Station, Fleming4 0 ton, N. J. Duplicate examinations were carried out on each sample and, - 0.8 in view of the close checks obtained, the results have been averaged in Table I. Preliminary experiments carried out on each of the corn samples, using the extraction method of Clark 1 and Gring, gave results ranging from 2 to 8 per cent lower than those reported in Table I. This discrepancy between methods may be explained by the fact that the esterified form of cryptoxanthin is less soluble in methanol than the free alcohol form and therefore complete extraction of this carotenoid would be more difficult in the method described by Clark and Gring. Since cryptoxanthin is less efficient than p-carotene as a source of vitamin A, the total quantity of active carotenoids in yellow corn does not necessarily designate the actual vitamin A activity of the corn, if figured on a &carotene basis. A comparison of the average results reported in Table I indicates that the provitamin A content of different samples of commercial yellow corn varies rather widely. This variation is influenced by the surface area of the kernels, which probably accounts for the greater quantity of provitamin A in the sample of Argentine yellow corn. The kernel size in this sample was considerably smaller, thus increasing the total surface or endosperm area in relation to the weight, leading to a relatively higher content of provitamin A per gram.
c
I
Chromatographic Separation of Carotene and Cryptoxanthin KO attempt has been made in this investigation to purify the 90 per cent methanol-soluble fraction (xanthophylls). Twenty-five milliliters of the concentrated carotene-cryptoxanthin solution were, however, chromatographed on a modified Tswett column and the zones developed by washing with purified heptane. Calcium carbonate (c. P. powdered), prepared by heating for 10 hours a t 200" to 300" C. in the absence of oxygen and then cooling in an atmosphere of nitrogen gas, was used as the adsorbent. The apparatus used for these experiments will be described in detail in another paper. Experiments conducted on pure /3-carotene obtained from the S. M. A. Corporation, Cleveland, Ohio, and repurified in this laboratory (m. p. 184" C. corrected, and optically inactive) and on purified cryptoxanthin (m. p. 168.6-169" C., and optically inactive) obtained chromatographically from yellow corn indicate that the carotene passes quantitatively through the adsorption column and into the filtrate, whereas the cryptoxanthin is adsorbed quantitatively by the activated calcium carbonate. The carotene fraction was readsorbed on a column of calcium hydroxide and proved to be homogeneous. TABLEI. CAROTENOIDS IN YELLOW CORN Sample
Carotene Plus Cryptoxanthin Carotene
Reid's Yellow Dent (Indiana) Argentine yellow corn Reid's Yellow Dent (Ohio) Yellow corn (New Jersey) Yellow corn (Indiana)
Carotene Cryptoxanthin
%
%
0.85 0.70
14.4 7.5
85.6 92.5
4.8
0.56
11.6
88.4
6.9
0.48
6.9
93.1
4.2
0.34
8.0
92.0
Gamma/g.
5.9 9.3
Gamma/g.
From the absorption curvesin Figure 1,photographed with a Bausch and Lomb medium-sized quartz spectrophotometer, i t is apparent that it is not possible to distinguish spectrophotometrically between carotene and cryptoxanthin when they occur simultaneously. The extinction coefficient, a t 4520 A. for purified cryptoxanthin in heptane, as calculated from the absorption curve in Figure 1, is 2430. By determining the intensities of absorption at 4500 A. before and after separating the cryptoxanthin from the carotene
Summary A simple rapid spectrophotometric method is described for determining the carotene-cryptoxanthin content of yellow corn. A chromatographic method is outlined, whereby the carotene can be separated quantitatively from cryptoxanthin. The results obtained on five commercial samples of yellow corn indicate that the carotene-cryptoxanthin contents vary considerably. A typical absorption curve for the cryptoxanthin-carotene fraction of yellow corn is reported, with a maximum a t 4500 A, Absorption curves for pure P-carotene and purified cryptoxanthin in heptane are given. A new extinction coefficient for pure cryptoxanthin in heptane is reported. Acknowledgment The author wishes to express his appreciation to H. B. Colman and L. T. Rosenberg for their helpful criticisms and assistance. Literature Cited (1) Buxton, L. O., and Dombrow, B. A., IND. ENG.CHEM., Anal. Ed., 10, 262-3 (1938). (2) Clark, G. L., and Gring, J. L., Ibid., 9, 271-4 (1937). (3) Coward, K. H., Biochem. J., 17, 145-56 (1923). (4) Karrer, P., Salomon, H., and Wehrli, H., Helv. Chim.Acta, 12, 790 (1929). (5) Kuhn, R., and Grundmann, C., Ber., 67, 593-6 (1934). (6) Steenbock, H.. Science,50,352-3 '(1919). RIDCEIVED November 17, 1938.
