V O L U M E 19, NO. 1, J A N U A R Y 1 9 4 7 Table X.
Analysis of Gasolines by the Lamp Method % Hydrogen
Sample By lamp method Aviation gasoline A 13.71,13.74 Aviation gasoline B 13.65,13.64 >Totor gasoline A 13.08,13.01,13.10 M o t o r gasoline B 13.10,13.09 * These samples were not analyzed
combustion Levin-chri$ modification 13.6 f 0.1
12.9
d 0.1
Approximate Composition Weight % P 0 N A 35 10 25 30 40 10 20 30 40 10 30 20 45 10 25 20
47 Determination of aromatic plus olefin by sulfuric acid a b s o r p tion Determination of H/C ratio of the saturate fraction directly and of the aromatic and olefin fraction by calculation 6. Determination of hydrogen content could be used to analyze mixtures of mono- and dicyclic aromatics. If rings of the phenylcyclohexane or phenylcyclopentane type are encountered, determination of the H / C ratio, followed by analytical hydrogenation, followed by another determination of H / C ratio, could be used t o indicate not only the aromatics, but also the weight per cent of naphthene ring. ACKNOWLEDGMENT
magnitude of the error in the determination of bromine number. By analyzing for per cent hydrogen (methylcyclohexene = 12.58% H , H / C = 1.7143, and heptene = 14.37% H, H / C = 2.0000),there should be no uncertainty. This technique may be applied to gasoline analysis: Determination of H / C ratio Removal of olefins by the Bond ( 2 ) method Determination of H / C ratio of olefin-free material 3. The H / C ratio may be used to measure the diolefin content of an olefin. The advantage of using this procedure is that conjugated or nonconjugated systems n-ould be equally amenable t o analysis. 4. Direct analysis for per cent hydrogen would probably be the most direct and most accurate method of following hydrogenation and dehydrogenation reactions. 5. The usual POSA analysis might be varied in this manner: Determination of H / C ratio
The authors wish bo thank Jack Grider for assistance with t h e analytical work, and Jack Hughes for his help in the calculation of some of the tables. LITERATURE CITED
Am. SOC.Testing Materials, Procedure ES-45a. Bond, G., IKD.EYG.CHEM.,ANAL.ED., 18, 692-6 (1946). Francis, A.. IND.ENG.CHEM.,35,448 (1943). Grosse. A. V., Refiner Natl. Gas M f r . , 18, No. 4, 149-57 (April 1939). Hindin, S. G., and Grosse, A . V., IND.ENG.CHEM.,ANAL.ED., 17, 767-9 (1945). Kurta, S.S.,and Headington, C. E., IND.ENQ.CHEM.,ANAL. ED.,9, 21 (1937). Kurtz, S. S., and Ward, A. W., Ibid., 10, 559 (1938). Kurta, S. S., and Ward, A. W., J. FranklinZn-st., 222, 563 (1936).
Analytical Methods for Carotenes of lycopersicon - Species and Strains F. P. ZSCHEILE'
AND
J. W. PORTER
Department of Agricultural Chemistry, Purdue University Agricultural Experiment Station, Lafayette, Znd. Studies on the nutritional importance of tomatoes have necessitated the development of rapid methods for the determination of carotenoids having provitamin A activity. Quantitative absorption curves of the principal carotenoids have in Lycopersicon species are presented. Spectroscopic methods for the determination of @-caroteneand lycopene are discussed in detail and results compared critically with those obtained by a chromatagraphic procedure. Sampling and extraction procedures are given and errors of recovery and those due to solvent impurity and carotenoid isomerization are discussed from the viewpoint of practical analysis. The proper application of spectrophotometric methods offers a valuable tool for the rapid analysis of tomato carotenoids.
T
H E spectrophotometric analysis of tomato fruit carotenes has been of considerable importance in a program conducted a t this station for the development of improved strains of tomatoes, genetically constituted to contain much more p-carotene(pr0vitamin A) than the present commercial varieties ( 4 ) . This problem will assume greater significance as the nutritional importance of tomatoes in our national economy becomes more generally recognized and as efforts t o improve the vitamin content of tomatoes begin to materialize in commercial production. It is also important in the study of fundamental problems of carotenoid physiology in relation to pigment development in plants. The complexity of the pigment system has made the analytical problem considerably more difficult than is indicated by earlier methods applied to mixtures of (presumably) all-trans forms (6, 7 ) , partially because of the presence of cis-isomers in some tomatoes (11) and in part because of the presence of addi1 Present address, College of Agriculture, University of California, Davis, Calif.
tional pigments (IO). However, from the practical viewpoint, after the various components have been identified, the spectroscopic analytical situation with regard to content of 8-carotene in the presence of lycopene does not appear so difficult as first suggested by other workers (6). EXPERIMENTAL
The following procedure has been designed to be as brief and simple as possible, so that it can be applied in a routine manner to hundreds of fresh and cannrd samplcs per season. Sampling. Fresh unpeeled fruits are used. If the fruit and core are large, the core is removed before cutting the fruit into pieces and homogenizing in a Waring Blendor. After about 2 minutes the fruits will be blended into a thick liquid mass which may be accurately sampled by pouring. Extraction and Separation of Carotenoids. A 20-gram sample, weighed to the nearest 0.1 gram, is poured into a clean Blendor cup and 75 ml. of acetone are added, some of which may be used t o rinse the sample from the weighing vessel. Sixty milliliters of hexane (b.p. 65-67" C.) are added and the mixture is blended
48 for 2 minutes. If the fruits are small, a* is characteristic of several species, enough whole fruits to make approximately 20 grams are blended directly in the solvent mixture. Two phases usually separate rapidly and the blended mixture is filtered on a Buchner funnel. The solid residue left on the filter paper is washed with a few milliliters of acetone, and then a small amount of hexane. Rarely, except with canned samples, will the residue (other than skin flakes) retain a n appreciable amount of pigments. Such samples are more completely extracted by shredding the filter cake and paper, blending 2 minutes with 50 ml. of acetone, and refiltering. All filtrates are combined in a 250-ml. separatory funnel. If the hypophase is colorless, it is drained and discarded: otherwise water is added gently before draining. The hyperphase is Tyashed carefully (to avoid emulsions) three times with water. A t the last washing any small amount of emulsion is discarded, as its pigment content will be negligible. Carotenols and chlorophyll are separated from the carotenes by immiscible solvent extraction. The hexane solution is washed once with 20 mI. of 90yo aqueous methanol (0.5 minute). All washings are performed 3000 40 42 44 4600 . 40 50 59 5400 by rotating the separatory fuiinels meWave Length, A. chanically. After clearing of the phase-: (15 minutes to 0.5 hour) the hypophase Figure 1. Absorption Curves of Six Carotenes Found in Fruit of Lycopersicon is drawn off and the hexane phase is Species and Strains (Solvent Hexane) washed with 20 ml. of 20% potassium hydroxide in mct,hanol (1minute). When Crosses indicate all-trans lycopene data from this laboratory clear (maximum 0.5 hour) the hypophase is discarded and another washing v i t h 20 ml. of 90% methanol is made (0.5 minute). After drawing off the hypophase, the carotene solution log (4875 5.) is washed vigorously three times with large quantities of n-ater. The funnel jtem is dried before drawing the hexane solution into I a 100-ml. volumetric flask. After rinsing the separatory funnel log -0 (4375 8.) I with hexane, the carotene solution is made to volume and placed in the refrigerator until analyzed spectroscopically. These solutions are usually bright and clear and seldom need to be dried The ratio for lycopene (0.94) is nearly the same as for p-caroover sodium sulfate. tene (0.88). Deviations indicate the presence of appreciable Spectroscopic Analysis. .i photoelectric spectrophotometer quantities of carotenes other than the aforementioned two. employing a large Muller-Hilger Gniversal double monochroR h e n such samples are encountered it is necessary to chromatograph an aliquot to determine the p-carotene content more accumator with crystal quartz optics is used. I t s performance has been described (16, rately. Chromatographic Analysis. K h e n results of greater accuracy Wave length 4875 A. is used in determining total carotenes for @-caroteneare needed, the carotene fraction 120 mIJ is chro(predominantly lycopene, neolycopene A, 8-carotene, and neomatographed on a column ((magnesia-Super Cel, 50-50)) 4 cm. @-carotene B). -4t this wave length the absorption values of long and 16 mm. in internal diameter (adsorptive powdered magall-trans @-carotene and lycopene are nearly equal (180 and nesia Yo. 2641, Westvaco Chlorine Products Corp., Sewark, approximately 183, respectively). A value of 181 for the specific Calif.). After adsorption of the carotene, the column is washed absorption coefficient has been found most reliable for mixtures with a small amount of hexane and then with 1 0 5 acetone which contain small amounts of cis-isomers of these twoopigments. in hexane until thrl 3-carotene is completely eluted. The eluate For the lycopene determination wave length 5020 9.is used. is washed twice with \vatclr, made to 25-ml. volunitb. and The specific absorption coefficient used for the lycopene fraction analyzed spectroscopically at 1360 and 4780 .\. ( 1 ’ . is 279 and the difierence between this value and the corresponding value of 42 for the 8-carotene fraction is 237. The 8-carotene fraction content is then obtained by difference. DISCUSSION Equations for the total carotene content and the per cent lycopene are as follows: Sampling. In prcJliminary studieh, scctions and equatorial slices From Beer’s law ( I ) , of ripe, large fruitb (Indiana Baltimore) were analyzed for carotene. h variation of 25 to 5 0 7 was found. Carotene contents of Total carotene (in micrograms per gram) = sections from fruits which appeared equally ripe differed by ISTO; 1 slices from corresponding fruits differed by 40%. Ellis and Hamlog f ( 4 8 7 d . ) X dilution factor X lo5 ner (3)have reported greater precision in their analyses of median 181 X length of cell (em.) x sample weight (grams) slices, but their error is still greater than that found when several fruits are blended. The use of whole fruits reduces the precision error to about *See 1 log f ( 5 0 2 0 i ) X 181 Extraction and Separation of Carotenoids. The skin pigments -___ - 12 of tomatoes are not completely extracted by acetone and hexane, I log 2(4875 K.) but are removed with hot alkali. Their absorption curves are not I x 100 Per cent lycopene = characteristic of carotenoids and presumably they can be ignored 237 from the provitamin X standpoint. They are alkali-soluble and cannot be transferred to hexane. I n work involving diverse types of fruits, it is advisable to deThe srymration of rarotcnoli and chloroph~-llfrom 8-carottme termine the absorption ratio
It).
V O L U M E 1 9 , NO. 1, J A N U A R Y 1 9 4 7
49
arc present in appreciable quantities i n only a fev strains of Lycopersicon. 01Carotene occurs very rarely. Keo-0carotene I: has been observed only in ranned samples in the authors' experience. Those special cases require different analytical treatment and can be detected by the study of relative absorption values a t appropriate wave lengths or by // I >BE A-CAROTENE U(1'31 chromatography. , In Figure 1 some discrepancies are evident between the writers' data for alltrans lycopene and corresponding data irom Zechmeister's work (12). Absorption values for several preparations of l>-copene diff&ed by only 1 to 2 7 , , but I h c spectr: of all preparations n'ere shifted 10 to 20 A. toward the blue with respect to Zechmeister's curve. Wave-length drum errors were eliminated by use of I I I I , IC->--+ , thcx mercury arc, but the possibility res3800 40 49 44 4600 48 50 59 51 mains that the solutions were slightly isomWave Length, k. c-rized, though great care was exercised to Figure 2. .hbsorption Curves of Four Additional Carotenes Found in Fruit avoid isomerization. Values a t the cis-peak of Lycopersicon Species and Strains (Solvent Hexane) agreed very well with Zechmeister's valuc: of 26 (12). -4 relatively - large - u11ct-rcould probal>ly be accomplished by chromatography of the hexane taint\- ( +6.4! h) in the absorption coefficients a t the highei;t. solution after washing it free of acetone. However, this methotl maxima for all-trans lycopcne was noted by Zechmeister et nl. might involve considerable difficulties in the determination o i (1.2). These comparisons iiidicate the extreme difficulty in pr('both 0-carotene and lycopene in a tomato extract. The latter paring solutions of pure lycopene in the all-trans form and it i q pigment is of considerable importance from the consumers' standwell known that this carotrnoid isomerizes in solution verv point, even though it has no vitamin A activity. ddaptation l i t rapidly. Moreover, the absorption changes in isomerization are the chromatographic method to routine analysis of a wide variety more marked with lycopene than with most other carotrnoitls. of tomato fruit types ~vouldprobably require more experience to I n Figure 3 are presented together the curves for tlir, pigmenta avoid serious losses than would be necevary in the us(' of inin-hich occur abundantly in Lycopersicon fruits f'i,oni r h c majority miscible solvent extraction methods. of sources and therefore are of most analytical iiitc~i~ost.8-CaroSeither alkaline methanol solution (10 t o 2OC; 1iiit:tsainm tc'ne and lycopene and t h c isomers i i i c ~ l r i c l c ~i lll Fixiirc. :
