Purity and Stability of Commercial Carotene Texas Agricultural Experiment Station, College Station, Tex.
G. S. FRAPS A N D A . R. KEMMERER
AROTENE, an important commercial product, is sold in considerable quantities for medicinal purposes, for adding vitamin A potency to animal Feeds, as a standard for vitamin A, for preparing reagents and standardizing photoelectric or visual colorimeters in the determination of carotene, and for other purposes. It is readily obtainable in crystalline or amorphous forms or dissolved in an oil. The composition, stability, and purification needed are of importance both from a scientific and a practical standpoint. The stability of carotene dissolved in oils, and in other solvents has been studied (1, 4), but little work has been, done on solid carotene.
C
Composition
Commercial carotene can be purchased In sealed glass tubes which have been evacuated. Analyses of one sample of such carotene were reported by Miller, ZscheiLe, and Hogness (6), who stated that it contains 6 per cent colorless impurities, 81 per cent 0-carotene, and 13 per cent a-carotene. Several samples of commercial carotene were analyzed for colorless impurities by colorimetric analysis of weighed portions. Chromatographic analyses were also made. Ten milligrams of the carotene were dissolved! in 1 ml. of chloroform and diluted to 100 ml. with petroleum ether. A 5-ml. aliquot was diluted to 250 ml., and the amount of pigment determined by the photoelectric colorimeter was expressed as carotene. The difference between the quantity found colorimetrically and the quantity weighed out was taken as the colorless impurity. This is not strictly correct, since some of the pigment was not carotene. Another 5-ml- aliquot was placed on a column of U. S. P. light magnesium oxide in a tube of approximately 8-mm. diameter, and the column was washed until the pigments had formed distinctive bands. The different bands of pigment were removed from the column and eluted with petroleum ether containing 2 per cent ethyl alcohol. The color was read and calculated as carotene. The average loss of pigment in the column was 5 per cent. The greatest loss experienced was 12 per cent. In calculating the percentage of constituents, the total amount of pigment recovered was used as 100. Analyses of several samples of solid carotcae freshly opened are given in Tables II and III. They contained 5 to 15 per •cent colorless impurities, 0.8 to 3.9 per cent colored impurities, 13.0 to 17.2 per cent a-carotene, and 80.5 t o 86.0 per cent ,0-carotene.
their color. At refrigerator temperature the carotenes lost 3 to 45 per cent of their color in a week. In 4 weeks of storage at refrigerator temperatures, very little carotene was lost from samples 2 and 4, and over 50 per cent was lost from samples 1 and 3. This is rather surprising but perhaps can be explained by the differences in physical or chemical properties of the carotenes. When they were examined under a microscope, carotenes 1 and 3 proved to be amorphous, and carotenes 2 and 4 to be crystalline. The amorphous material would have a greater surface exposed than the crystalline material and would consequently be oxidized at a more rapid rate. Also, on subsequent analyses, carotenes 1 and 3 were found to contain 15 per cent cf noncolored materials, and carotenes 2 and 4 only 6 and 5 per cent, respectively (Table I). These impurities might in some way facilitate the oxidation of carotene. The fresh and exposed carotenes were analyzed chromatographically, and the results are given in Table II. The freshly opened samples of carotene contained 0.8 to 3.9 per cent of colored impurities, while the samples exposed to air at room temperature for 4 weeks contained 0.6 to 28.6 per cent of colored impurities. Colored impurities may thus develop in carotene during exposure to air. The samples of amorphous carotene contained and developed greater amounts of colored impurities than did the crystalline carotenes. T A B L E I.
E P P B C T OP EXPOSURE TO A I R ON THE COMPOSITION OF COMMERCIAL CAROTENE Colorless,
Carotene 1 Freshly opened Room temperature 1 week 2 weeks 4 weeks Refrigerator temperature 1 week 2 weeks 4 weeks Carotene 2 Freshly opened Room temperature 1 week 2 weeks Refrigerator temperature 1 week 2 weeks 4 weeks Carotene 3 Freshly opened Room temperature 1 week 2 weeks 4 weeks Refrigerator temperature 1 week 2 weeks 4 weeks Carotene 4 Freshly opened Room temperature 1 week 2 weeks 4 weeks Refrigerator temperature 1 week 2 weeks 4 weeks Purified carotene in petroleum ether Room temperature 1 week 2 weeks 4 weeks 8 weeks Refrigerator temperature 1 week 2 weeks 4 weeks 8 weeks
Deterioration of Carotene Exposed to A i r
When the sealed tubes are opened audi the solid carotene is exposed to the air, it decomposes with the formation of both colorless compounds and colored compounds, as shown by the following experiment: Samples of carotene, manufactured by different concerns, were stored at refrigerator temperature and at room temperature in contact with air. At the end of various periods the quantity of carotene was determined. Weighed amounts were dissolved in petroleum ether, and the color of the solution was read by a KWSZ colorimeter. The relative quantities of colored substances were determined by the chromatographic mesthod described above. For purposes of comparison, a solution, of carotene in petroleum ether was stored at the same time. The results are given in Table I. Int 1 week at room temperature the samples of solid carotene lost 10 to 62 per cent of 846
%
CHEMICAL
Impurity Colored, % of total color
15
3.3
62 75 96
5.5 6.3 18.4
45 55 78
4.3 6.2 10.4
6
3.9
28 56
1.4 2.0
6 6 3
1.0 0.7 0.8
15
2.3
53 85 99
6.6 14.0 28.6
24 43 57
4.3 5.5 7.1
5
0.8
10 18 55
0.5 1.6 1.7
3 1 —3
0.7 0.6 0.6
12 22 42 87
... ... ... ... ... ... ...
5 8 14 34
August 10, 1941
N E W S
Purification Carotene used in this laboratory is purified (3) by the following method: 0.1 gram of carotene i s dissolved in 2 ml. of chloroform and precipitated with 25 ml. of methanol. The precipitated carotene is filtered off, washed with about 1 ml. of methanol, and dried in a vacuum desiccator over calcium chloride. This method of purification was applied to carotene which had been exposed to the air, with the results given in Table II. TABLE II. EFFECT OF PURIFICATION ON COLORLESS AND COLORED IMPURITIES IN SOLID CAROTENE Colored ImpurColorless ity, % of Total Impurity, % Color Before After Before After purifipurifipurifipurifi cation cation cation cation Carotene 1 Freshly opened 15 0 3.3 1.6 2 weeks at room temperature 75 22 6.3 5.3 2 weeks at refrigerator temperature 55 10 6.2 3.6 Carotene 2 Freshly opened 6 0 3.9 3.3 2 weeks at room temperature 56 1 2.0 0.7 β 2 weeks at refrigerator temperature 0 0.7 0.3 Carotene 3 Freshly opened 15 3 2.3 1.3 2 weeks at room temperature 85 37 14.0 3.4 Carotene 4 Freshly opened 5 1 0.8 0.9 2 weeks at room temperature 18 0 1.6 1.4
The purification removed practically ail the colorless im purity from the freshly opened samples except carotene 3 in which 3 per cent impurity was left after purification. With the samples stored 2 weeks a t room temperature, the purifica tion reduced both the colorless and the colored impurities. Samples 2 and 4 were appreciably better than samples 1 or 3 . The effect of the purification on the proportions of a- and βcarotene is shown in Table III. Although i n some cases puri fication increased the percentage of 0-carotene and decreased that of α-carotene, the changes were not large and were smaller than could be expected. T A B L E I I I . E F F E C T OF PURIFICATION ON P E R C E N T A G E S OF aAND B-CAROTENE IN TOTAL COLORED CONSTITUENTS B-Carotene. % a-Carotene, % Before After Before After purifipurifipurifi purification cation cation cation Carotene 1 Freshly opened 13.8 11.9 83.0 86.5 2 weeks at room temperature 10.4 11.8 83.3 83.0 2 weeks at refrigerator temperature 12.4 14.6 81.7 81.7 Carotene 2 Freshly opened 13.0 13.6 83.6 83.0 2 weeks at room temperature 8.3 13.5 90.0 85.7 2 weeks at refrigerator temperature 13.2 13.4 86.1 86.4 Carotene 3 Freshly opened 17.2 15.2 80.5 83.5 15.8 17.4 70.2 79.0 After 2 weeks at room temperature Carotene 4 Freshly opened 13.3 14.7 86.0 84.4 13.7 13.3 84.9 85.2 After 2 weeks at room temperature
E D I T I O N
847
impurities in 4 weeks at room temperature. The deteriora tion of some samples of carotene is small. Purification of deteriorated carotene by solution in chloro form and precipitation with methanol reduces the percentage of colorless compounds considerably in carotene of poor quality, or in case of carotene of good quality, to 0-1 per cent. The colored impurities are also reduced to 0.3-3.3 per cent in carotene of good quality, or 1.3-5.3 per cent in carotene of poorer quality. Carotene of poor quality after exposure to the air for several weeks was not purified sufficiently for use as a quantitative standard. Literature Cited (1) Baumann, C. Α., and Steenbock, H., J. Biol. Chem., 101, 561 (1933). (2) Carter, C. P., and Gillam, A. E., Biochem. J., 33, 1325 (1939). (3) Fraps, G. S., and Kemmerer, A. R., J. Assoc. Official Agr. Chem., 22, 190 (1939). (4) Fraps, G. S., and Kemmerer, A. R., Texas Agr. Expt. Sta., Bull. 557 (1937). (5) Gillam, A. E., El Ridi, M. S., and Kon, S. K „ Biochem. J., 31, 1605(1937). (6) Kraybill, H. R., and Shrewsbury, C. L., J. Nutrition, 1 1 , 104, footnote (1936). (7) Zechmeister, L., and Tuzson, P., Biochem. J., 32, 1305 (1938). PRESENTED before the Division of Agricultural and Food Chemistry at the 101st Meeting of the AMERICAN CHEMICAL SOCIETY, St. Louis, Mo., April 7
to 11, 1941.
Redesign of Soda Pop Bottles Cuts Costs TRANSPORTATION costs and storage space for bottled soft drinks can be cut in half by redesigning bottles to fit closely in vertical racks, according to Walter H. Bell, industrial design student in the Cooper Union Art Schools, New York. Without altering outside dimensions of the 5-cent soda bottles in use today, Mr. Bell has designed a bottle which reduces the size of a case of 24 from 1824 to 909 cubic inches. The bottles, which have oval bottoms and flat, tapering sides, may be laid like building blocks, and carried in a light-weight rack. Carrying capacity of a standard three-axle truck can be doubled by using the new 24-bottle vertical case, and the storage space required in bottling plants for empty cases is only two fifths that for wooden ones. The vertical racks reduce by 0.5 ton the weight of a 300- case load. The shape of the bottles makes it possible for an ordinary bottle-washing machine to cleanse 20 at a time instead of the present 16. Because outside measurements have not been changed, minimum alterations are needed to adjust machinery to new shape bottles.
According to Gillam and co-workers (5) adsorbents may convert a-and b-carotene into isomers, but Zechmeister and Tuzson (7) and Carter and Gillam (2) found that this con version takes place in solution and is not due t o adsorbents, the speed of the reaction depending on the temperature. At the boiling temperature of the solvent, this conversion is claimed to take place rapidly until equilibrium is reached. Summary Commercial carotene purchased in sealed tubes may con tain 5 to 15 per cent of colorless impurities. The colored material may contain about 13 per cent a-carotene, 83 per cent B-carotene, and 1 to 4 per cent colored impurities. Carotene exposed to the air may deteriorate rapidly, with a loss of as much as 62 per cent colored material at room tem perature and 45 per cent at refrigerator temperature in one week, and the production of as much as 2 9 per cent colored
Comparison of oval-shaped bottles in carrying case at right and ordinary case at left emphasizes the space saved by redesign.
