INDUSTRIAL AND ENGINEERING CHEMISTRY
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Vol. 36, No. 4
cuprous oxide-zinc oxide paint, and a control pigmented only with barytes were used. The formulations are described in FormulaCompn.* a‘t. % Table IV. The panel preparation schedule is shown in Table V, tion No. Pigmentation Vehicle Pigment Vehicle together with the inspection results after 4-month immersion a t a Zn dust, ZnO 25- a1 1/1 tung/linseed 71.5 28.5 ptenolio varnish Daytona Beach. b Znasbestine yellow, TiOz, Same 67.0 33.0 The presence of tIyo barrier coats separating the antifouling c Iron oxide Same 40.0 60.0 paiht from the metal surface appears to have substantially elimd Blue lead, ZnO, 33- a1 dehvdratedcastor 6 0 . 0 40..0 inated any accelerated corrosion a t the scribe marks, although asbestine, celite pf~enolic~varnish e Red lead 25-ga1. tung phenolic 7 0 . 0 30.0 the steel was initially laid bare a t these marks as in the previous varnish I C u powder, barytes 8-gal. 1/1 t m d 1 i n - d 38.5 Cu content experiments. Although the relation is not clear-cut, it seems oumar varnish likely that, as the thickness of the barrier layer is increased, there CUZO,ZnO Same 38.0 cu content Barytes Same 75.0 23.0 is a rapid decrease in the tendency t o accelerate corrosion localls a t exDosed areas of steel substrate. These findings confirm best shipyard practiceTABLE Jr. FOULIXG US. C O R R O S I O X AFTER MONTH IMI\fERSION namely, the use of multiple barrier coats, or of Antivery heavy undercoats, beneath antifouling paints. fouling Over-all 2nd Panel 1st Corrosion Coat Coat Fouling NO. Coat Scribe marksa P i t ratingb They further suggest that thick, relatively impera C 241 Slight 6 s-m meable barrier coats, topped by medium coats of a 242 C Slight; deep a t one spot 6+ m antifouling paints, should give better corrosion prob C 243 Slight but varying 4 f m b C Occasional deep sections 7f s 244 tection coupled with efficient antifouling action C C Slight b u t varying 4 m 245 C c Slight 4 m 246 than can be had with light priming coats and a C C 247 Slight 5+ heavy antifouling layer. This point is currently C Slight 248 C IO 249 Vs., few small pits 9+ OS f under investigation. d C 9 250 Slight 9 us
TABLE ITT. PAINT COMPOSITIONS
a
---
:
251 252
.
e
e
C C
r”
8
9
Fairly uniformly etched Fairly uniformly etched
3
5
s m-d
LITERATURE CITED
(1) Evans, U. R., “Metallic Corrosion, Passivity and 0 Average depths were less than 1 mil, with spotty variations from 0 t o 5 mils. There v,as Protection”, pp. 513-35, London, Edward Arnold no significant relation between composition and depth of attack. b There were too few pits to allow valid measurements: ratings are on a 0-10 scale, with & Co., 1937; LaQue and Cox, Proc. Am. SOC. depth indicated as s (shallow), m (medium), and d (deep). Testing Materials, 40, 670-89 (1940) ; Wesley, Z b i d . 40, 690-io4 (1940). (2) LaQue, F. L., Gibson Island Symposium on Corrosion, 1912; private communications. INFLUEYCE O F INTERMEDIATE BARRIER COATS (3) Young, G . H., and eo-workers, IND.ENG.CHEM., 35, 432. 436 (1943). A4notherseries of saribed test panels was exposed which carried (4) young, G , H., and co-workers, unpublidlled data. ,
a variety of undercoat permutations involving five different barrier paint,s For the antifouling compositions a copper paint, a
CoxTRIBvTIox from the Multiple Fellowship on protective coatings of Stoner-Mudge, I ~ c . ,a t ~ \ i e i i o nInstitute.
Carotenoids in Corn By- products W. Baumgarten, J. C. Bauernfeind C. S. Boruff H I R A M WALKER & SONS, INC., PEORIA, ILL.
N ANIMAL nutrition certain carosenoids serve as precursors of vitamin -4;others not possessing vitamin activity are mainly of interest because of their pigmenting characteristics. Steenbock (16) noted that vitamin potency was associated with the yellow color of plants, possibly carotene, and demonstrated that yellow corn was a better source of provitamin A than white corn. At present (14) nine or more pigments are reported to possess vitamin A activity. Previously i t was reported (3, 10,11) that the carotenoid pigments present in egg yolk and in the body fat of the fowl consist almost entirely of xanthophylls with small amounts of cryptoxanthol and carotene, whereas cattle deposit carotene primarily in their body fat and in the fat of their milk. Corn distillers’ by-products are used primarily in dairy rations (7) as desirable ingredients, high in total digestible nutrients, although during the past few years they have been recognized as significant sources of the water-soluble vitamins in the rations of poultry and swine (6, 16). These corn distillers’
by-products are made from yeast-fermented grain mixtures in which yellow corn predominates, a grain regarded as an important source of the carotenoids in the feeding of farm animals. I n view of these facts an investigation was initiated to determine the carotenoid content of distillers’ by-products. By-products used in this investigation have been defined by the Association of American Feed Control Officials (1). Briefly i t may be stated that corn distillers’ dried grains with solubles is the total dried stillage containing both the spent grain residues and the thin stillage; corn distillers’ dried grains is the spent grain residues or screenings; corn distillers’ dried solubles, as the name suggests, is the dried thin stillage or solubles. Usually six samples of each product were analyzed, each representing a t least 20 tons or more of the product. Hybrid dent corn (1-2 years old) was analyzed for comparison. EXTRACTION AND CHROMATOGRAPHIC TECHNIQUE
Samples of distillers’ by-products or of yellow corn were continuously extracted in a Soxhlet apparatus with Skellysolve B (65.5-70.5’ C.) in the dark until the effluent solvent was colorless by visual inspection. It was soon discovered that two or three times the amount of carotenoid pigment could be
April, 1944
INDUSTRIAL AND ENGINEERING CHEMISTRY
345
extracted if a previous alcoholic saponification of the samples was employed. The following extraction method was therefore adopted. The sample (100 grams) was refluxed with alcoholic potassium hydroxide (290 ml. of 95% ethanol plus 10 grams potassium hydroxide) for one hour on the water bath. After cooling, the saponified mixture was filtered through a single-thickness paper thimble and the residue extracted in a Soxhlet apparatus with Skellysolve B in the dark. The alcoholic alkaline filtrate was diluted with an e ual volume of water and exhaustively extracted with Skellyso%e B. All Skellysolve B extracts were combined and washed free of alkali and ethanol with water in a separatory funnel. The first water washing was re-extracted with Skellysolve B, and the fraction returned to the principal extract. The washed Skellysolve B extracts were dried by contact with anhydrous sodium sulfate and concentrated in vacuum. Completeness of extraction was tested by subjecting the residue to a second saponification. bands formed were removed from the column and individually eluted with a methanol-Skellysolve B mixture. . The carotenol fractions were readsorbed on calcium hydroxide (325-mesh, Marblehead Lime Company), and the bands were eluted. The concentration of the eluted pigments was measured in a Coleman Universal spectrophotometer at the wave length of 450 my. Since all the igments were not obtainable in pure crystalline form, crystaiine p-carotene (s.M.A. Corporation) was used as a reference standard. The wave band width of the Coleman instrument is approximately 32.5 mp. Some error may appear in the quantitative evaluation of carotenoids having the maximum of absorption outside the limits of the wave band employed. The extinction coefficient, E: (450mp.) of the @-carotenestandard was determined as 1780 on the Coleman Universal spectrophotometer and 2480 on the Beckman spectrophotometer. The absorption characteristics of all the carotenoids were measured in purified Skellysolve B with a Beckman spectrophotometer. Their absorption curves, plotted in Figures 2 to 6,are expressed in the same optical density range (log Io/Z) for comparative purposes. The authors departed from the customary method of carotenoid separation (1A) in that they dispensed with partition between immiscible solvents entirely and chromatographed the Skellysolve B extract direct. Partition between immiscible solvents was excluded as preliminary studies on repeated washing of the Skellysolve B extract with 90% methanol seemed to remove a small amount of cryptoxanthol (5Y0 or less) while a small amount of zeaxanthol (2-7%) remained in the Skellysolve B extract, as judged by the position of the pigments in a chromatographic analysis of the two fractions. IDENTIFICATION
Figure 1. Chromatograms of Corn Distillers’ Dried Solubles (/eft) and Yellow Corn (right)
The Skellysolve B extract containing all carotenoids was directly introduced on the ignited alumina column (Merck’s reagent grade) and washed with small volumes of Skellysolve B. The chromatogram was developed with benzene (Merck’s reagent grade) in 1-2 hours. After the column was removed, the individual pigment bands were separated and immediately eluted with a solution containing 98 parts of Skellysolve B and 2 parts of methanol. Benzene with methanol was used to elute the more strongly adsorbed pigments. With careful elution an average of 95y0 of the pigments introduced on the column was recovered. Hurried elutions resulted in lower recoveries. The carotene fractions of distillers’ by-products and yellow corn were subjected to further adsorption. The carotene fraction in Skellysolve B is placed on a column of activated alumina (aOO-mesh, Alorco), and the chromatogram is developed with chbroform (Merck’s reagent or Baker’s C.P. grade). The
OF CAROTENOIDS
The chromatograms of corn distillers’ by-products showed four distinct pigment bands; those of yellow corn showed three (Figure 1). Zeaxanthol was isolated by Karrer, Salomon, and Wehrli (4) and Kuhn and Grundmann (6). The latter workers isolated cryptoxanthol, and showed by chromatographic analysis on activated alumina that zeaxanthol is strongly adsorbed on top of the column, that cryptoxanthol locates itself near the center, and that p-carotene proceeds to the bottom of the column. Pigment bands C1 and C2 of yellow corn were identified as zeaxanthol and cryptoxanthol by their position on the ignited alumina column. Mixed chromatograms of bands D1 with C1 and D2 with C2 revealed them to be identical. The absorption curves of bands D1 and D2 of distillers’ by-products are shown in Figure 2. Their absorption maxima are manifested at 442 and 468 with a shelf of 422 mp and compare closely. The absorption characteristics of the two purified carotenols were recently determined by Zscheile, White, Beadle, and Roach (19); however the use of different solvents does not permit close comparison with our data. Bands D1 and D2 of distillers’ by-products could be further resolved on calcium hydroxide with Skellysolve B. It was revealed that the zeaxanthol fraction contained 8% neozeaxanthol (upper baFd) and the cryptoxanthol fraction, 23% neocryptoxanthol (lower band). Fraps and Kemmerer (8) showed that both cryptoxanthol and neocrypto-
INDUSTRIAL A N D E N G I N E E R I N G CHEMISTRY
346
xanthol possess comparable biological actitivy and hence separation for the biological evaluation is unnecessary. Chromatographic treatment of bands Dl and D2 on activated alumina using Skellysolve B, benzene, or chloroform did not effect a separation. The nonidentity of bands D3 and C3 are shown by mixed chromatography and by absorption spectra. Since cryptoxanthol and neocryptoxanthol are not separated on ignited alumina but on calcium hydroxide, it follows that D3 cannot be neocryptoxanthol. Originally we postulated that D3 might be neocryptoxanthol in view of its position on the column. Band D3 has a maximum of absorption a t 444 and 472, a minimum
a t 463, and a shelf at 422 mp. A similar pigment, with maxima a t 474, 445, and 425 and a minimum a t 463 mp, whose position of adsorption is below cryptoxanthol, is described by White, Zscheile, and Brunson (18) and is assumed by them to be a monohydroxy-a-carotene. Band D3 is designated in our paper as the unknown carotenoid. The identity of bands D4 and C3 was established by mixed chromatography. Their absorption spectra are compared in Figure 3 and are similar. When these pigments are chromatographed with 8-carotene on ignited alumina with benzene, a single band is formed. This would lead the observer to believe that pigment bands D4 and C3 are &carotene; however, this
/
380
400
390
430
420
410
440
450
460
470
Vol. 36, No. 4
490 370
480
380
BAND C3b
--- BAND
Mb
390 400 410 420 430 4 4 0 WAVELENGTH IN M ILL IM IC RONS
450
460
470
. FIGURE
Figure 2. Absorption Spectra of Noncarotene Pigment Bands of Corn Distillers’ By-products i n Skellysolve B
--
4
Figure 3. Absorption Spectra of p-Carotene and Carotene Pigment Band of Corn Distillers’ By-products and Corn i n Skellysolve B
2
-
Figure 4. Absorption Spectra of Fraction a of Carotene Pigment Band of C o r n Distillers’ By-products and Corn in Skellysolve B Figure 5. Absorption Spectra of Fraction b oficarotene Pigment Band of Corn Distillers’ By-products and Corn i n Skellysolve B
I
370
i
380
I
l
l
l
1
1
1
1
1
1
1
1
390 400 410 420 430 440 WAVELENGTH IN MILLIMICRONS
1
1
450
1
1
460
1
1
470
1
_
Figure 6. Absorption Spectra of Fraction e of Carotene Pigment Band of Corn Distillers’ By-products a n d Corn i n Skellysolve B
April, 1944
INDUSTRIAL A N D ENGINEERING CHEMISTRY
observation is not substantiated by absorption data. Bands D4 and C3 could be resolved on activated alumina with chloroform into three components. Bands D4a and D3a were on top of the column and formed a narrow orange zone. Bands D4b and C3b constituted the main fraction and formed broad yellow zones. Bands D4c and C3c, light yellow zones, passed quickly through the column and usually were collected in the filtrate. The similarity of the components of the carotene fraction of distillers’ by-products and corn was established by mixed chromatograms using activated alumina. Their absorption spectra are plotted in Figures 4, 5, and 6. Band C3a has absorption maxima a t 448, 425, 401, and 379 mp. Band D4a has maxima a t 451, 426, and 402 mp. Its absorption below 380 mp was not investigated. Unnamed carotene I, described by White, Zscheile, and Brunson, has similar absorption characteristics and is possibly identical with band C3a. They did not mention the position of unnamed carotene I on the column relative to that of p-carotene but compared it with similar pigments described in the literature. Fraps and Kemmerer (2) reported a new carotene, K carotene, which was adsorbed above p-carotene on the column and had similar absorotion characteristics.
Mixed chromatograms help sustain this claim. The similarity of and bands D4c and c3c is demonstrated by absorption observations on their adsorption characteristics; however, their identity is unknown. QUANTITATIVE DATA ON CAROTENOID CONTENT
Quantitative data on the carotenoid fraction of distillers’ by-products are included in Table I ; the zeaxanthol and unknown carotenoid content are similar for all products. Corn distilled dried solubles contained slightly less cryptoxanthol than the other two by-products. The greatest difference was in their carotene content; corn distillers’ dried solubles contained the least, corn distillers’ dried grains, the most, and corn distillers’ dried grains with solubles, a n intermediate amount. Table I contains data on the carotdnoid content of yellow corn for comparison. Since it was shown that the carotene fraction of corn distillers’ by-products and yellow corn contain carotenes other than @-carotene, Table I1 was included for comparison of quantitative data on the resolution of the carotene fractions. I n all cases half or more of the carotene fraction was made up of 8carotene, one fifth was probably identical with unnamed carotene I of White et al. (18), and the remaining fifth was an unidentified carotene. Data on relative quantities of components in the carotene fraction of yellow corn are interesting. The amount of p-carotene decreased if the carotene fraction was stored in the refrigerator prior to separation as compared to immediate separation. This suggesta that the other two components may be derived from @-carotene. On the basis of the carotenoid content in yellow corn and through the use of by-product recovery figures, one would expect to find approximately 17-21 micrograms of eeaxanthol, 4-5 micrograms of cryptoxanthol, and 2-3 micrograms per gram of carotene in the composite by-product, corn distillers’ dried grains with solubles. Actually only one third to one half of the expec.ted eeaxanthol and carotene was found. The actual and expected cryptoxanthol values agree reasonably well. ‘ During the fermentation process and by-product recovery, heat is applied in the cookers, stills, evaporators, and drum dryers which no doubt contributes to the partial destruction of the carotenoids. Nagy (9) states that the carotenoids of corn are fairly resistant to oxidation, as indicated by the persistence of the yellow color of gluten during processing. However, he reports the presence of isomerized carotenoid pigments in gluten. No quantitative data are presented.
341
TABLE I. CAROTENOID CONTENT OF CORNDISTILLERS’ BY-PRODUCTS AND YELLOW CORN Product Corn distillers’ dried solubles Corn distillers’ dried grains with solubles
Corn in Mash
Carotenoid Fractions, Microgramq’Gram
84 65
Zeaxanthol 8.8 6.5
Cryptoxanthol 3.9 3.7
84 05
8.3
7.6
5.6 5.0
’
%a
Unknown carotenoid Carotene 0.7 0.8 0.8 0.6
0.8 0.8
1.2 1.0
Corn distillers’ dried grains 80 8.0 5.1 0.8 1.8 Yellow corn 8.5 1.8 0.0 1.1 a Contained 10% barley malt and some rye grain: rye grain was shown to possess a low carotene and total oarotenoid content (8).
..
TABLE 11. RESOLUTION OF CAROTENE PIGMENT BANDOF CORN DISTILLERS’BY-PRODUCTS AND YELLOWCORN Product
8:;solubles :;E;!:$
dd,:i;;2ti;ith
Fraction a 19.0 21.0
Fraction b 57.8 63.0
Fraotion e 23.2 15.4
18.6 54.8 20.6 21.7 62.7 15.6 28.0 49.6 22.4 ily pre ared carotene solution. ;ene soktion after 9-day storage in a refrigerator.
$Yellow ~ f ~ corn , “ i Ba ~~~~&dried
\
Carotenoid values obtained in this study for agree fairly well with values published by Fraps and Kemmerer and by peterson,Hughes, and payne F~~~~and Kemmerer obtained carotenoid values by the partition method and by a chromatographic technique developed by them. Peterson, Hughes, and Payne determined carotenoid content by a modification (12) of the Guilbert method whereby carotenoid separation is accomplished by partition between immis,cible solvents. Recent reports (2, 17‘) demonstrate the wide variation that exists in the carotenoid content of yellow corn. ACKNOWLEDGMENT
Acknowledgment is made to the Analytical Division of the Northern Regional Research Laboratory, for the use of their facilities t o obtain absorption spectra. LITERATURE CITED
(1) Assoo. of Am, Feed Control Officials,Ofloial Pub., 1942, 31. (1A) Assoc. of 05cial Agr. Chem., Official and Tentative Methods of Analysis, p. 369 (1940). (2) Fraps, G. S., and Kemmerer, A. R., IND.ENQ.CHEW,ANAL. ED., 13, 806 (1941). (3) Gillam, A. E., and Heilbron, I. M., Biochm. J.,29, 1064 (1935). (4) Kai-rer, P., Salomon, H., and Wehrli, H., Helv. Chim. Acta, 12, 790 (1929). ( 6 ) Kuhn, R., and Grundmann, C., Ber., 67, 593 (1934). (6) Krider, J. L.,Fairbanks, B. W., and Carroll, W. E., J . Anima2 Sci.. 1. 359 11942). (7) Morrison, F. B:, “Feeds and Feeding”, 20th ed., p. 399,Ithaca, Morrison Pub. Co., 1936. (8) Murri, I. K., Soviet Plant 2nd. Record, 4 , 125 (1940). (9) Nagy, D.,Iowa State Coll. J. Sci., 15, 89 (1940). (10) Palmer, L. S.,J . Biol. Chem., 23, 261 (1915). Palmer, L. S., and Eckles, C. H., J . Biol. Chem., 17, 191, 211 (1914). Peterson, W. J., Hughes, J. S., and Freeman, H. F., IND. ENG. CHEM.,ANAL.ED., 9,71 (1937). Peterson, W. J., Hughes, J. S., and Payne, L. F., Kansas AQ. Expt. Sta., Tech. Bull. 46 (1939). Rosenberg, H. R., “Chemistry and Physiology of Vitamins”, p. 39 (1942). (15) Sloan, H.J., Poultry Sci., 20, 83 (1941). (16) Steenbook. H..and Boutwell. P. W., J. Biol. Chent., 41, 81 (1920). (17) White, J. W., Brunson, A. M., and Zscheile, F. P., IND. ENQ. CHEM.,ANAL.ED., 14, 798 (1942). (18) White, J. W.,Zscheile, F. P., and Brunson, A. M., J . Am. Chem. SOC.,64,2603 (1942). (19) Zscheile, F. P., White, J. W., Beadle, B. W., and Roach, J. R., Plant Phyaiol., 17, 331 (1942). P R B S E N T ~before D the Division of Biological Chemistry at the 104th Meeting of the AMERICANCEE~MICAL BocImy, ButIalo. N. Y
