V O L U M E 1 9 , NO. 8
614 spectrophotometric study and to thank I. Weber and I. Geld for their assistance in obtaining the experimental data. LITERATURE CITED
(1) Hague, J. L., and Bright, H. A,, J . Research *YaatZ.Bur. Standards,
26, 405-13 (1941). (2) Lundell, G. E. F., Hoffman, J. I., and Bright, H. A., “Chemical
Analysis of Iron and Steel,” p. 214, S e w York, John Wiley & Sons, 1931. (3) U. S. Steel Corp. Chemists, “Sampling and Analysis of Carbon and iilloy Steels,” p. 92, New York, Rheinhold Publishing Corp., 1938. THEopinions expressed are those of the authors and are not to be construed as reflecting the official views of the Xavy Department, through whose permission this article is published.
Carotenoids of Stored Dehydrated Carrots GORDON AIACKINNEY AND W. E. FRATZKE Division of Food Technology, University of California, Berkeley, Calif. A procedure has been devised for determining whether significant changes occur in the relative proportions of the carotenoid pigments of dehydrated carrots on storage. Present methods of carotenoid assay are ill adapted to accurate determination of all components where large numbers of samples are involved. A critical analysis of spectrophotometric data indicates no major change in the
I
N ADDITION to a- and p-carotenes, the carrot contains numerous carotenoids present in minor proportions. One frequently needs to know whether significant changes have occurred in the relative abundance of these components in processed carrots on storage in order, ultimately, to assess the effect of a given processing treatment, and this paper is concerned with methods that are applicable. h detailed study of the absorption spectra of crude extracts and of the chromatographed components, in the range 510 to 310 mp, is reported. MATERIALS
Five samples of Imperator carrots were selected, four of which had been in storage for years or more, under unfavorable conditions. The initial carotene content of all samples ranged from 90 to 100 mg. per 100 gram of dry weight, and four samples had lost from 40 to 80% of their original endowment. Significant trends should therefore be discernible. The samples were prepared as follows: 1. Sulfited and dehydrated in the laboratory, winter of 1942, stored (a) a t room temperature, (b) for six months a t 48“ C., in air, in air-tight jars. Sample l a was light in color, sample l b a dark brown. 2. Dehydrated in the factory in 1943, (a) sulfited, (b) control; in air a t room temperature. 3. A 5-gallon can, commercial pack, in nitrogen a t room temperature. PREPARATION OF EXTRACT
Samples are passed through a Wiley mill, 60-mesh, and used immediately, unless otherwise noted. Sormally a 1- to 2-gram sample is taken, covered with 50 ml. of acetone, stirred, decanted, and washed two to three times with small portions of acetone. At this stage, about 80% of the carotene has been removed. The residue is then covered with water and stirred gently to ensure absorption, and the acetone treatment is continued until the residue is colorless. The combined filtrates, about 200 ml., are then transferred to petroleum ether which is washed and dried in the usual way, and made to 50- or 100-ml. volume. Twenty minutes suffice for this operation. One half is then chromatographed directly, and an aliquot of the remainder is diluted for direct measurement of the optical density. It has been found more satisfactory to rehydrate the sample after a preliminary acetone extraction. If no water is added, prolonged grinding is necessary. If it is added a t the start, minute carrot particles will form a faint emulsion, and lower values are obtained. CHROVATOGRAMS
Micron-brand magnesia and Hyflo-Supercel were used for adsorption (3). A column filled to a height of 15 to 20 cm., 2.2 cm. in diameter, was used. The chromatogram was developed with petroleum ether containing 3y0 acetone.
proportions of cis-trans isomers on storage. Oxidation of phytofluene and r-carotene certainly proceeds no faster than that of other components. This is of interest in view of their marked instability on extraction. Products or processes for which significant anomalies in spectrophotometric data are found would, in addition, require chromatographic examination.
The pigments of the stored samples were fractionated as follows: 1. Phytofluene, discovered by Zechmeister and Sandoval (5, 6), with maxima in petroleum ether about 368, 347, and 330 mp. (Since the relative heights of the bands are identical, differences of 1 mp or less in the positions are ascribed to solvent.) 2. a-Carotene, maxima about 475 and 445 nip. 3. p-Carotene, maxima about 480 and 450 mp. 4. r-Carotene described and provisionally named by Strain (3) and by Xash and Zscheile ( 2 ) , maxima about 425, 400, and 375 mp. 5 . 7-Carotene, maxima about 490, 460, and 435 mp. 6. hliscellaneous minor components, including xanthophylls, eluted from the column with acetone and transferred to petroleum ether. In all cases where acetone had been used, even in eluates from the mixed solvent, it was removed by washing with water, prior to measurements of optical densities, made a t suitable dilutions, with a Beckman quartz spectrophotometer and a tungsten light source. The petroleum ether containing 3% acetone gives excellent and rapid separation of the a- and p-carotenes with a colorless eluate betaeen them. The effectiveness of this solvent is limited to the first five components mentioned above, which pass into the eluate within 2 hours. Beyond this point, additional washing is ineffective. Shorter columns, while effectively separating the CY- and p-carotenes, do not permit clean-cut separation of the last of the 8-zone from the p- and y-zones. The latter was normally not estimated because it represented an insignificant fraction of the total. I n fact, spectroscopic identification of the y-carotene required 5 to 10 grams of carrot powder. Severtheless, the band served as a useful reference marker on the Tsn-ett column. The r-carotene was considerably more abundant, and was routinely estimated. I t occupies an intermediate position on the column between the p- and y-zones and is pale yellow in color. Three problems are presented: 1. The proportion of the original that can be accounted for by the separated components. 2. The homogeneity of a given zone. 3. Changes in the proportions of the various components. RECOVERY O F PIG.MEIITS
The first problem was to determine whether adequate recovery of chromatographed material could be obtained. Four wave lengths were selected: 450, 445, 400, and 345 mp. The last two were chosen because of the influence of carotenoids with maxima in the’ near ultraviolet. Two crude extracts 11-ere prepared, and
AUGUST 1947
615
one aliquot of each was chromatographed, while one was diluted for direct measurement. Four separate portions were collected a t convenient time intervals, vithout regard to the component passing into the eluate a t the time of collection. A fifth portion was obtained by elution of all remaining components with acetone, then transferred to petroleum ether. Results are shown in Table I, the sum of the portions being given in row 6 for comparison with the original, row 7. ,411 values were calculated to the same concentration. Over 90cc of the absorption can be accounted for a t these wave lengths, ewept sample 2 a t 400 mp, where only847, was recovered. This may be due to poor reproducibility in a region of rapidly changing absorption, or to loss of a component with relatively higher absorption in this region. T o obtain the reproducibility shon-n in Table I, all measurements had to be made on the same day, as rapidlj- as possible.
The data are not inconsistent with those of Kemmerer and Fraps (1). Calculating the percentage of their crude carotene, recovered as CY- and @-components (Table 11, page 456) we find for 0, 2, 6, 10, and 14 months' storage, 97, 86, 91.3, 87, and 89.7%, respectively. Since their petroleum ether extracts are first partitioned with 90% methanol, their base is more selective than the authors', which includes all the carotenoids present in the carrot. Table 111. Sample
Carotenoids in Stored Carrot Samples
Phytofluene
a-Carotene
@-Carotene
?-Carotene
M g . p e r I00 orams d r y wezght
HOMOGENEITY OF ZONES
T o ensure that each zone was spectroscopically homogeneous, a series of measurements was made on successive fractions from both a- and p-carotene zones. I n the original experiments, late in 1945, the results appeared anomalous, but xvere explicable in terms of the discovery of phytofluene (6, 6) and inadequate separation on columns of 8- to 12-em. length of y-carotene, {-carotene, and p-carotene where the last-mentioned was overwhelmingly predominant. The anomaly caused the authors to use the longer columns. Even here it Tvas occasionally necessary to readsorb the combined y- and {-carotene zones to eliminate the last trace of the p-component. A short column, approximately 7 em. long, suficed for this. An example of the homogeneity of the p-carotene zone is shon-n in Table 11, which indicates, for the visible range, the time required for the first and last portions to pass into the eluate. The constancy of the ratio for the tn-o samples a t each wave length indicate. pigment identity. CHANGES IN COMPONEhTS
When the pon-dered (60-mesh) samples are stored, deterioration is rapid. Thus within three months comparable extracts from samples l a and l b declined from 0.201 and 0.126 to 0.01s and 0.070, respectively. However, ratios for absorption remained constant-e.g., 450/480 mp, 1.21, 1.22, 1.21, 1.24; 4451425, 1.23, 1.28, 1.19, 1.21; 4451475, 1.14, 1.15, 1.13, 1.17. Similar results were obtained with samples 2a and 2b. The results indicate that the chromatographed zone is spectroscopically homogeneous, and that there has been no significant change in the evtracts in the proportions of such cis-trans isomers as were present originally. Table I.
Chromatographic Recoveries, Optical Density
mfi
Samole Portion
1 2 3 4 5
Sum Orlginal
0 0 0 0 0 1 1
450 1 245 0 471 0 534 0 157 0 1 0 528 1 58 1
2 015 309 040 915 176 455 57
0 0 0 0 0 1 1
445 1 263 0 469 0 517 0 155 0 125 E 529 1 57 1
2 017 335 044 889 2 457 57
0 0 0 0
400 1 106 0 176 0 193 0 058 0 0 639 0 707 0
e 0 0
2 012 128 021 324 0 605 715
0 0
0
0 0 0 0
345 1 076 0 051 0 042 0 014 0 068 0 251 0 253 0
Table 11. Homogeneity of a @-CaroteneZone Wave Length, mfi
500 485 480 475 470 460 450 445 440 430 420 410 400
First Portion 0.173 0.563 0.628
(0.628) 0.598 0.661 0 742 0 712 0.638 0,550 0.500 0.361 0,26i
Last Portion 0,272 0.878 0.980 0 955 0.920 1.06 1.17 1.11 0,990
0.882 0,778 0.564 0.408
Ratio 1.57 1.56 1.56 (1.52) 1.54 1.60 1.58 1.56 1.55 1.55 1.55 1.56 1.53
2 042 057 021 068
072 260 264
ESTIMATION OF INDIVIDUAL COMPONENTS
The phytofluene is obtainable in the eluate ahead of the acarotene. However, a t least the first half of the eluate of the latter contains phytofluene. I t is simplest therefore to collect the two together and to treat them as a 2-component mixture. The p-carotene presents no difficulty, but it is better to collect the r-carotene separately than to estimate it in a mixture with the p-carotene when the proportions are so different. The y-carotene is a minor component with negligible absorption. The final fraction, removed from the column with acetone, accounts for about 15% of the absorpt,ion in the visible, and this percentage increases slightly on storage. The absorption coefficients, in liters per gram centimeter, used for computing the results of Table I11 Tvere: phytofluene 120 a t 347 and * O at 445 mp; a-carotene 10 a t 347 and 255 a t 446 mp; p-carotene 249 a t 450 mp; {-carotene 200 a t 426 mp (8). h sample of crystalline a-carotene prepared in this laboratory gave a ratio of Eat?to Elas of 0.049, in agreement iTith a value calculated from curves presented by Zechmeister and PolgAr (4). For an equal mixture by weight of a-carotene and phytofluene, an error of 8% (10 in 120) is introduced unless a correction is made for the former. In carrots, the correction is essential. Loss of 207, of the carotene in a carrot is accompanied by, or parallels, production of off-flavors that render the food unacceptable. Four of the samples discussed here had deteriorated far beyond this limit; yet the changes in the proportions (Table 111) are relatively small, compared with the over-all loss of pigment in the stored samples. I t is believed, therefore, t,hat the effectiveness of a given processing treatment can be adequately assessed by measurements on crude extracts, a t suitable time intervals. For dehydrated carrots treated as in present commercial pract,ice, measurements a t one wave length should be adequate, but if measurements are made at, 425, 400, and 365 mp in addition to wave lengths for masima for a- and 8-carotenes, the influence of phytofluene and similar compounds is included. An additional safeguard against change is thus ohtaincd. ' LITERATURE CITED
(1) K e m m e r e r , h. R., a n d Fraps, G. S., I n d . Eng. Chem., 38, 457 (1946). (2) N a s h , H. A . , and Zscheile, F. P., Arch. Biochem., 7, 305 (1945). (3) S t r a i n , H. H., J . Bid. Chem., 127, 191 (1939). (4) Zechmeister, L., and Polgbr, A , , J . Am. Chum. Soc., 65, 1522 (1943). ( 5 ) Zechmeister, L., a n d S a n d o v a l , A., Arch. Biochem., 8 , 425 (1945). (6) Z e c h m e i s t e r , L.. and Sandoval, A., J . Am. Chem. SOC.,68, 197 (1946).
PRESESTED before the Division of .Igricultural and Food Chemistry a t the 110th Meeting of the AJIERIC.ANCHEJIICALSOCIETY,Chicago, Ill. The subject matter of this paper has been studied in cooperation with the Committee on Food Research of the Q M Food and Container Institute for the Armed Forces. T h e opinions and conclusions are the authors' and d o not necessarily reflect the views or endorsement o i the War Department.
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