September, 1944
INDUSTRIAL AND ENGINEERING CHEMISTRY
Thus it appears that the best available values for the differences in entropy or in the free energy function between isomers are still those published by the writer (IO) in 1940; the absolute value of the heat capacity of branched paraffins not yet studied experimentally may best be assumed to be the same as the normal isomer. In particular there is no need to revise the calculations of isomerization equilibria made by Rossini, Prosen, and the writer (16). No attempt is made in this brief note to compare these results with the numerous empirical or semitheoretical calculations which have been published nor with old experimental values where more accurate ones exist. The recent papers of Stull and Mayfield (IS) give many references to such work. ACKNOWLEDGMENT
The results of the present paper are being combined with recent heat of combustion values of Prosen and Rossini for the normal paraffins to give free energies of formation and related data, in connection with Research Project 44 of the American Petroleum Institute, a t the National Bureau of Standards. The writer wishes to thank E. J. R. Prosen for carrying out some of the calculations here presented.
CAROTENECONT
831
LITERATURE CITED (1) Aston, J. G . , and Messerly, G . H., J. A m . Chem. SOC.,62, 1917 (1940). (2) Birge, R. T., Rea. Modern Phys., 13, 233 (1941). (3) Dailey, B. P., and Felsing, W. A., J . Am. Chem. Soc., 65, 42, 44 (1943). (4) Dennison, D. M., Rev. Modern Phys., 12, 175 (1840). (5) Eucken, A., and Sarstedt, B., Z. physik. Chem., B50, 143 (1941). (6) Kemp, J. D., and Egan, C. J., J. Am. Chem. Soc., 60,1521 (1938). (7) Kistiakowsky and Rice, J . Chem. Phys., 8 , 610 (1940). ( 8 ) Leininger, R. F., unpublished measurements, Univ. Calif. (9) Messerly, G . H., and Kennedy, R. M., J . Am. Chem. SOC.,62, 2988 (1940). (10) Pitzer, K. S., Chem. Rev., 27, 39 (1940); J . Chem. Phys., 8 , 711 (1940). (11) Pitzer, K. S., J . A m . Chem. Sac., 62, 1224 (1940). (12) Ihid., 63,2413 (1941). (13) Pitzer, K. S., J. Chem. Phys., 12, 310 (1944). (14) Pitzer and Scott, J. Am. Chem. SOC.,63, 2419 (1941).
(15) Rossini, F. D., Prosen, E. J. R., and Pitaer, K. S., J. Research Natl. Bur. Standards, 27, 529 (1941). (16) Sohumann, S. C., Aston, J. G . , and Sagenkahn, M., J . A m . Chem. Soc., 64, 1039 (1942). (17) Stitt, F., J . Chem. Phys., 1, 297 (1939). (18) Stull, D. R., and Mayfield, F. D., IND.ENG.C H ~ M35, . , 639, 1303 (1943). (19) Witt, R. K., and Kemp, J. D., J.Am. Chem. SOC.,59, 273 (19371,
ALFALFA
etention on Dehvdration and Storage J
Various treatments are reported which stabilize the carotene in alfalfa during the process of drying. Blanching the fresh alfalfa with steam, prior to drying, furnishes complete protection for the carotene, and considerable protection is afforded when fresh ground alfalfa was treated with certain chemicals before it was dried. Two types of chemicals are used-namely, antioxidants and substances which are known to inactivate enzymes. Diphenylamine and hydroquinone are the most effective of the first type; thiourea and sodium cyanide are more effective than any other substances tested of the second type. The carotene content of alfalfa meal decreases as the temperature of storage is increased. However, essentially no change has been noted in the carotene content of alfalfa meals stored at 3" C.
M
ORE and more attention is being directed toward the
retention of natural nutritional constituenta in feeds as well as in foods since the practice of fortifying them with various food concentrates has come into common use. The primary purpose of this study on the carotene content of alfalfa is to find a practicable method to stabilize this precursor of vitamin A during dehydration and storage. The alfalfa used was stripped from the plants in the field. I n this way, principally leaves were collected since the bulk of the stems were avoided, and more nearly uniform samples were available for analysis. Stripping the plants has an additional advantage in that the carotene content of the material is much greater than if the whole plant is used, and thus the effect of a particular treatment can be noted more readily. The dehydration of the various samples was carried out in a Freas circulating air oven a t 65" C. The fresh and blanched samples were placed on towels on trays so that contact with metal
RALPH E. SILKER, W. G. SCHRENK, AND H. €1. ICING Kansas Agricultural Experiment Station, Manhattan, Kans,
was avoided. (The effect of certain metals will be reported in B later paper.) The dehydrated alfalfa was ground in a Wiley mill using a 2-mm. mesh screen; samples were taken for analysis, and the remainder was stored in glass jars with tight-fitting covers. A greater loss of carotene accompanies this slow method of dehydration than that of the rapid commercial dehydrators, but the different samples of a given experiment which are compared were subjected to the sbme conditions. METHOD OF ANALYSIS
The method used for det,ermination of carotene was essentially that of Wall and Kelley ( I S ) , which is a modification of the method of Moore and Ely (IO). Duplicate samples were run except for a short period when help was not available. Five grams of the fresh material (stemmy portions were broken into small pieces to avoid excessive splashing when the Blendor was started) was placed in a Waring Blendor with 60 ml. of a 1-1 Skellysolve B-alcohol mixture, and sufficient alcohol was added to form a foaming mixture. The moisture content of the alfalfa determines the amount of alcohol needed; 125-150 ml. were usually required. Blending was carried out for 10 minutes, and a nearly colorless pulp remained on filtration. The Blendor and pulp were carefully washed with Skellysolve B, and the extract and washings were treated with 100 ml. of water to remove most of the alcohol and cause the Skellysolve B layer to separate. Addition of a small amount of anhydrous sodium sulfate to the extract eliminated the formation of an emulsion. After separa-
INDUSTRIAL, AND ENGINEERING CHEMISTRY
832
Vol. 36, No. 9
A dehydrated sample, usually 1 gram, was reconstituted by allowing it to stand in a small beaker with 5 to 7.5 ml. of water for 10 minutes before blending. The volume of alcohol needed to form a foaming mixture was usually 90 to 100 ml. The method gave satisfactory checks and avoided, to a large degree, the isomerization of &carotene t o the “neo-p-carotene” of Beadle and Zscheile (2). The method of calculating the percentage of isomerization of p-carotene assumes the presence of only two pigments, and neglec‘ts, of course, the presence of the additional isomer which Kemmerer and Fraps ( 7 ) reported since this study was undertaken. The carotene values obtained when refluxing is used are usually mmewhat higher than those with other methods unless special precautions are taken (17) This is due to the presence of noncarotenoid pigments which Moore (9), Fraps and Kemmerer (6), Wiseman et at. (ZO), and others have Figure 1. Effect of Blanching, Dehydration, arid Storage on pointed out. Passing the carotene extract obCarotene Content of Alfalfa tained by the Peterson, Hughes, and Freeman F = fresh; FB = fresh blanched; D = dehydrated; A = blanched; 0 = control. A, B , C stored at 3’ C.; D stored at room temperature. method (13) through a magnesia column gave values that compared favorably with those reported here, but the percentage of p-carotene was usually slightly lower TABLEI. EFFECTOF BLANCHING ON CAROTENE CONTENTOF ( 1 7 ) . Thevalues obtained with soda lime as adsorbent, accordALFALFA ing to Kernohan @), were also high since some of the xanthoTotal Carotene, Mg./lOO G . Alfalfa (Dry Wt.) phylls were eluted with the carotene. A simplified method, Sample Blanching Fresh, Fresh Blanched which avoids refluxing or the use of a Waring Blendor in exNo. Time, Min. Fresh dehydrated blanohld riehydrateh tracting carotene from dehydrated samples, has been described 300 0’ 45.3 .. .. .. 4710 40:6 300 5 (17). 456 0 43.7 34.4 .. Moisture samples were taken at the time of the carotene de456 10 .. .. 42:3 termination so that all results are reported on a dry-weight basis. 53.0 .. 405 0 I t was most convenient t o use a n air oven at 105’ C.; although .. .. 55 8 55:8 405 5 ., ~. 55.5 53.7 405* 5 the values were not the same as if a vacuum oven had been used, results are comparable. 321 0 39.0 16.8 I .
321 321
7 10
.. ..
..
..
45:7
436 436
0 7
48.0
28.6
..
438
0 7
43ag**
*
471 0 471 7 Ground after blanching.
..
.. .. .. 23.7 44.0 .. .. 17.8 ,. 30.3 .. .. .. ** Ground before blanching.
39:2 42.9 43:9
I . .
I
36:4
25:o
tion, the water layer was extracted three times with 35-ml. portions of Skellysolve B. The combined extracts were then concentrated on a steam cone, dried with anhydrous sodium sulfate, and finally drawn through a containing a 2 to 1 mixture of Hyflo Super-Ce nesia (Micron brand 2641) to separate the carotene from the chlorophylls and xanthophylls. Heating is one of the methods which Pol& and Zechmeister (14) used to isomerize p-carotene, but this change was largely avoided if the carotene extracts were not concentrated below 30-40 ml. The columns, approximately 75 mm. in length, were prepared by adding the adsorption mixture under suction. A layer of anhydrous sodium sulfate (about 1 cm.) was placed above the adsorbent, and the column washed with Skellysolve B before the extract was poured in. A column may be used for several separations if it is carefully.washed with Skellysolve between samples. The carotene was eluted from the column with a 401,acetone-Skellysolve B mixture. The eluate was made up to a volume of 250 ml. (100 ml. for the low-carotene samples), and the optical densities were measured a t 436 mp and 478 mp with a Beckman ( 3 ) spectrophotometer. The two readings were made so t h a t it would be possible to calculate the percentage of p-carotene according t o the method of Beadle and Zscheile (9).
EFFECT OF BLANCHING
The value of blanching certain vegetables with hot water before home canning has been recognized for many years. The advent of quick frozen and dehydrated foods has been responsible for much research on the length of time, on various techniques,
L
I
STORAGE TIME IN MOFlTHS
Figure 2. Effect of Grindipg, Dehydration, Chemical Treatment, and Storage at 3 C. on Carotene Content of Alfalfa F = fresh; G = fresh ground; D = dehydrated; A = treated; 0 = control. A = thiourea; B = sodium cyanide; C = diphenylamine; D = hydroquinone
INDUSTRIAL AND E N G I N E E R I N G CHEMISTRY
September, 1944
ABL LE 11. EFFECT OF GRINDING AND DEHYDRATION OK CAROTENE COXTENT OF ALFALFA Fresh
Sample
Strippeh,
NO.
X1g.a
Fresh
Groun6, lIg.a
Loss on Frzbh DaGrindinq,
350 41.5 35.7 360 60.6 55.0 46.5 370 54.8 380 53.2 43.3 52.4 390 435 45.0 44:O 470 30.3 24.8 'z Total carotene, mg./100 granis
353
%
hydr;t$l,
7.6 15.2 20.8
Nolle 0.5 0.25
0.125 0.0625
352
None Diphenj-!amine Olive oil hiinersl oil
0. 5 0.6 0.5
211 6
None 0.5 0.23
23.7 33.2 33.1 33.2 31.6
0.123
0.0325
._
.. ..
,,
None Thiourea Thiourea Thiourea Thiouiw
433 433 433 438
.. .. .. ..
..
572 372 372 372 372
None
..
16.4
11.0
Sone 0.5 0.S 0.5
None Na cyanide Na cyanide N a cyanide Na cyanide
17,8 12.5
47:9 27.5 43.8 17.8 41.3 alfalfa (dry-weight basis).
None Urea Thiourea Acetamide
438
16.6
..
5L0
27:3
1O:O
13.3 9 0
17.8 28.4 27.8 25.7
.. .,
,
29.1
..
.. , .
12.5
.i ..
.. ..
11.2 119
.
.
.~ I
.
.. I
.
~I 1
,
,
.
.. .
Ground. DB- Loss on hydrated, D o h y d r a Alz," tion, $&
41.2
18.2
g53
332
01
tio?, 70
24.8 27.3
13.9
,353 353
332 332
L w
D:hpdra-
I
24.6 30.9 20.7 26.1
53.5 79.8 61.8 79.4
11.3
12.8
23.7
46:2
12,3
60.5
15.6 16.2 16.0 9.8 0.4 24.7 23.6 23.4 9.1 .. 9.7
15.6 9.5 23.4 9.6
1 3 . 1 17.
