Determination of Carotene in Alfalfa and Cereal Grasses H. L. VITCHELL
4YD
H. H. KING, Ziansns Agrirultztrnl Experiment Station. V n n h a t t a n , Zian.
The photochemical destruction of carotene in the presence of chlurophj11 w a s negligible during carotene analj ses under normal conditions of laborator? illumination. Destruction was appreciable, however, when the saniples were placeil directlj in sunlight or left unprotected for seieral hours on the laborator? table. A method of carotene analysis is proposed \+ hich eliminates the present laborious extraction procedure for remobing carotene from fresh plant tissue. It involves the inacti\ ation of carotene-destroying enzymes by blanching or autoclaiing, drjing at low temperatures, and the use of a simplified procedure for extraction of carotene from the dried material. The adTantages and disadTantapes of the method are discussed.
Fl’KOKITZ (3) has s11ow11 that suiilight may cause considerable destruction of carotene in solutions that also mitain chlorophyll. He reported that extracts of cooked vegetables lost more carotene than did extracts of raw vegetables \\.lien exposed to sunlight. His experiments, however, were not liesigned to shon- the amount of carotene that \ d l be destroyed (luring actual carotene analyses. I t is desirable that the extent i i f this loss he known. so that the need for prevent,ive niea:’ur~s niay be considered. This paper evaluates the photochemical destruction of carotene that occurs during actual carotene determinations in both fresh and dried plant tissue. I t is also concerned with a procedure tor eliminating thr use of the \Taring Blendor and for reducing sampling (war in the deterniination of carotene in fresh plant tissue. DESTRUCTION OF CAROTENE BY LIGHT
Five-grani samples of blanched (1 minute with steanii and unblanched fresh alfalfa leaves lvere analyzed bv the method of Kall and Kelley (j), except that) they were not”minced prior to comminution. The blanched samples n-ere included in the experiment because of the report by Pepkowitz that carotene destruction is greater i n extracts of cooked than ralv vegetables. Part of the samples n-ere comminuted in a Waring Blendor ancl rxtracted near the windon-s of the laboratory on a clear day. .Although the samples were not placed directly in the rays of thr sun, the laboratory table was brightly illuminated. During 2 hours between blending and chromatographing the samples were not protected from light. The remainder of the samples were Iic~lrlin a rrfrigerator until night, irlirn the!- xvcre coniniinntr~cl
Table I .
Effect of Sunlight on Determination of Carotene in Fresh and Dehydrated Alfalfa
Trra t men t
Carotene
> Unblanched leaves, dark Unblanched leal-es, suiiliglit Blanched leaves, d a r k Blanched leaves, sunlight Dehydrated meal, dark Dehydrated meal, sunlight
Destruction
8 . d r y ut.
848 793 877 850 965
258
c
... &.-I
i :1 2 , li
Table 11. Effect of Intensity and Type of Light on Destruction of Carotene in Extracts of Dehydrated ilfalfa \leal Exposure Tinie,‘ H o ii r.3 0
Carotene y . / g . d r y u’t. S o r n i a l Ish. illumination (day) 283 3 270 3 248 Direct sunliaht 0.25 194 Ultraviolet Iainp (1 foot) 0.5 286 100-watt m a z d a l a m p (3 feet) 1 279 ” Does not include exposure during eoncentration of solution.-. Treat nient
Lors durina Expo~ur~
5% 0
4.6 12.4 31,s 0
aiid anal!-zcd: the only light S U U I ’ C ~was a 25-watt IIazda laiiil) equipped n-ith a JVratten Series 0 pliotographic darkroom filt .As a further precaution. extractions were made in amber gl separatory funnels. The, wsults of the experiment are presentcd in Table I. The effect of sunlight 011 the tlc.terinination of carotene in dehydrated alfalfa meal b?- the. simplified procedure of Sillier. Schrenk, and King (4)was also s t u d i d because the length of expo~uroto sunlight is much Shoi,t~~r than x i t h other methods. Sixty milliliters of a 33y6 solution of acetone in Skellyholvi. B were added to 1-gram samples of meal. After standing in the dark overnight, the samples were filt,ered and washed \vith Skellysolve B on a Buchner funnel. The filtrates were trancfcrred to beakers and concentrated 011 a steam plate in a hood t o about 40 nil. The solutions were then chromatographed on a 1 to 2 mixture of magnesia (Micron brand No. 2641) and Suprrcel. The carotene was eluted from the adsorbent with a 4co solution of acetone in %kellysolve €3. Carotene concentration \\-as measured at 4360 A. with a Beckman spectrophotometer. The unprotected samples analyzed by this method were exposell to light for about 45 minutes, during the filtration and evaporat inn procedures. The samples which were protected from light \rtw analyzed at night, using the darkroom filter described ahow. The reaults of this experiment also appear in Table I. Using the method of Silker, Schrenk, and King with ali:+If:t nieal samples, the effect of qualit>-antl intensity of radiation 011 the photochemical destruction of carotene was studied. The t,xtracts after filtration wcre transferred to 600-ml. clear Pyrm beakers antl exposed to various type’s of radiation, as shown i l l Table 11. After exposure, the extracts were Concentrated anll chroniatographed without further protection from light, as the data of Table I indicate that little destruction occurs during the latter steps. I t is apparent from Table I that, under normal conditions of illumination in the laboratory, the destruction of carotene by sunlight was negligible during analysis. Table 11, however, slio\v.s I hat photochemical destruction i m s appreciable when extractr; w r c placed directly in the rays of the sun or left unprotccted for several hours on the laborator>-tahlc. Ultraviolet light caused no dwtruction during a 30-minute exposure. Light from a llazda lamp was not detrimental. Thus, it appears that precautions need to he ialien to prpvc,nt, photochemical destruction if sunshine falls directly upon the samples. 11111y Thii carotene value for a blanched sample (Table I) was somewhat higher than the value for the corresponding unblanrhed -ample. This confirms thc data of Zscheile and Khitmore (61, \ ~ h ofound that carotcne was more easily and more thoroughly cwractctl floin blanched than from unblanched alfalfa 1eavc.s. fi.bTIHATIO\ OF CAROTEYE 01.F R E S H PLANT T I S S U E
1 9
Onc 01 the most nidely used methods of extracting carotene ironi t i w l i plaut tissue iq that of \loore and Ely ( 2 ) , which con-
637
ANALYTICAL CHEMISTRY
638 Table 111.
Effect of Temperature and Time of Drying
(Comparison of Moore-Ely extraction procedure with a proposed method for determination of carotene in fresh alfalfa) Loss Method of Drying Drying by Extraction Temp. Time Carotene Drying _O Hours y/g. d r y wt. % hloore-Ely extraction ... , . 374 0
c.
Proposed method
Table IV. Sample
36 50 65 65 65 80 80 80 100 100
22 4 2.5 4 6 2 4 6 1 2
356 348 353 350 332 323 302 299 315 306
4.8 7.0 L 6
6.4 11.2 13.6 19.3 20.1 15.8 18.2
Determination of Carotene in Fresh Plant Tissue Rloore-Ely Extraction y per
Alfalfa 1 .4lfalfa 2 Wheat grass 1 Wheat grass 2 Bromegrass 1 Bromegrass 2 Oat grass a Dried a t 65. C. for 4 hours.
428 440 457 600 510 601 404
Proposed Method" gram d r y u e t g h t 403 42 1 501 601 238 ,766
400
sists of comminuting the sample in a \Taring Blendor with a mixture of ethanol and petroleum ether. There are certain disadvantages to this method as it now exists. Splashing during comminution may result in some loss of solution. I t is also necessary to subject the comminuted solution to a laborious extraction procedure to remove the carotene completely from the ethanol phase. For this reason, the number of determinations that one operator can perform in a day is small. Mitehell and Hauge ( 1 ) have reported that an autoclaved sample of alfalfa which was dried in a Despatch oven a t temperatures below 33" C. had lost essentially no carotene when analyzed by a method similar to that of Moore and Ely. The experiments described below 1% ere performed to show the feasibilitv of determining the carotene of fresh plant tissue by blanching or autoclaving to inactivate the carotene-destroying enzyme S,I stem, drying a t low temperatures, and extracting the carotene from the dried residue by the simplified procedure of Silker ef wl. Samples of whole alfalfa \%-erewrapped in towels, autoclaved for 5 minutes a t 5 pounds' pressure in a horizontal steam Qterilizer, and dried in a Despatch oven under various conditions of time and temperature. The dried samples were ground to pass through a 20-mesh screen and were analyzed bv the method of Silker, Schrenk, and King. Grinding to 2Gmesh has been found ade uate in this laboratory, although some workers prefer finer grin8ng (6). Ten 5-gram samples of the fresh material were also analyzed by the extraction procedure of 31oore and Ely as employed in the method of Wall and Kelley, to serve as a basis for comparison. Ten determinations were made because of the large sampling error that occurs with such a nonuniform material as fresh whole alfalfa. The data presented in Table I11 show that both time and temperature of drying affected the amount of carotene that was destroyed. A long drying period a t low temperature resulted in less destruction than a short drying period a t the higher temperatures. Long drying periods are to be avoided, however, because they increase materially the time required to complete the determination. Drying for 4 hours at 65" C. appears to be optimum, for a t higher temperatures the amount of destruction increased rapidly and became appreciable. With some samples, depending upon size of sample and type of plant material, the drying time cannot be reduced much below 4 hours. Inactivation of the carotene-destroying system of the plant tissue by blanching or autoclaving is an essential operation. Unless this is done, extensive carotene destruct ion will occur during
drying. I n conjunction with the experiment reported in Table 111, an unblanched sample was also dried a t 65" C. for 4 hours. Carotene destruction in this case amounted to 31.5%, compared to 6.47, for the blanched sample. The carotene content of a variety of fresh plant materials LYas determined by both the proposed method and the Moore-Ely extraction procedure. Values are presented in Table I V for comparative purposes. The chief disadvantage of the proposed method is the length of time required to complete the analysis. Because of the 4hour drying period and the necessity of allowing the sample to stand overnight in contact with the extracting solution, the determination cannot be completed in one day. However, during much of this time the analyst is free to carry on other work, so that the actual amount of time devoted to the determinatioii is comparable with that for other methods-it is estimated that about 80 minutes of actual working time are required to make a single determination by the proposed method, nhile about 90 minutes are needed by the Wall and Kelley procedure. The method will be of especial value to those who have a large number of fresh samples requiring attention at a given time and a limited amount of technical help to care for them. After blanching and drying, the samples can be held for several weeks a t freezing temperatures without loss of carotene. The proposed method greatlv reduces sampling error (Table V). By other methods the plants are finely chopped and mixed, and be&use the leaves contain much more carotene than the stems, slight variation in sampling the chopped tissue may result in exceedingly poor checks betxeen duplicates. Furthermore, the determination usually cannot be repeated if the analytical technique is faulty. With the proposed method, on the other hand, much more uniform mixing and sampling of the dried material can be achieved. There wlll also he a reserve supply of material available if it becomes necessary to repeat the determination because of analytical failures. The slight destruction of carotene that may occur during drying is a small price to pay for the increased precision that is obtained.
Table V.
Precision of Moore-Ely Extraction Procedure and Proposed Method .
Moore-Ely Extraction Individual Maximum analysis Av. variation Y/B.
d r y wt.
Alfalfa 360 360 433 446 457 470
Y/B.
Y/B.
Y
d r y uii.
d r y wt.
Y
110
376 379 383 383 384 388
382
12
Y/#.
d r y ut
421
Proposed Method Individual Maximum AV. variation analysis
Oat grass
LITERATURE CITED
(1) Mitchell, H. L., and Hauge, (1946).
S. M.,J. B i d . Chem.,
164, 543
(2) Moore, L. A., and Ely, R., IND.ENG.CHEM.,ANAL.ED., 13, 600 (1941). (3) Pepkowitz, L. P., J . Biol. Chem., 149, 466 (1943). (4) Silker, R. E., Schrenk, W. G., and King, H. H., IND.ENG.CHEM., .ANAL. ED.,16,513 (1944). ( 5 ) Wall, &I.E., and Kelley, E. G., I b i d . , 15, 18 (1943). (6) Zscheile, F. P., and Whitmore, R. A., I b i d . , 19, 170 (1947). RECEIVEDJune 30, 1947. Presented before t h e Division of Agricultural and Food Chemistry a t the Midwest Regional Meeting of the AMERICAN CKEWCALS O C I E T Y , Kansas Clty, hlo., June 23 t o 25, 1947. Contribution 334, D e p a r t m m t of Chemistry, Kansas State College. Work supported by the Kansas Industrial Development Commission.
