ANALYTICAL EDITION
March 15. 1943
Complete extraction of the starch is indicated by recovery experiments, the ability to duplicate results closely, and the check of results on samples reground and extracted with alcohol with those obtained directly determined without extraction. Other evidence is t h a t a reextraction of the residue on the filter paper after the extraction of starch gave a negligible starch content. A starch determination on niaterial left on a 40-rnesh screen after grinding the sample in the disintegrator and washing through the screen also gave a negligible result. Further evidence of the reliability of the method has been obtained in its application. A group of raw pea samples were graded for quality with a tenderometer and then analyzed for starch. Figure 2 shows the relationship between these two factors. Fifty samples of frozen lima beans Fere graded for quality organoleptically and by brine flotation. Starch analyses on these samples correlated very well with both the organoleptic and brine flotation grading. The relationship between proportion of sinkers in 20 per cent brine and percentage of starch in lima beans is shown as a n example in Figure 3.
Rapidity of the Method Starch in a single sample can be determined in 20 t o 30 minutes if t h e reagents have been previously prepared. Forty t o 50 samples can be analyzed for starch in a n 8-hour day if a routine is established.
Perchloric Acid Hazards There is no danger whatsoever from the 72 per cent perchloric acid itself, other than the usual hazards of strong
179
acids. The cold diluted acid is a very poor oxidizing agent, as is evidenced by the fact that it will not release iodine from iodide ion. Hot perchloric acid, however, forms unstable compounds with organic material and for this reason the treated samples should never be heated. Samples have been allowed to stand in the laboratory at room temperature and to dry on filter paper for several days without accident.
Summary A very rapid and reasonably accurate method for the determination of starch in certain vegetables has been developed. It includes grinding the fresh sample in a Waring Blendortype disintegrator, extracting the starch with 4.0 to 4.8 molal perchloric acid, and estimating b y photoelectric colorimeter the dissolved starch indicated b y the blue color produced with iodine. Alcohol extraction of t h e products studied was found to be unnecessary. The use of a red filter in the colorimeter considerably reduces the error produced by dextrins when present.
Literature Cited (1) Assoc. Official Agr. Chem., Official and Tentative Methods of Analysis, Chap. 27, p. 360, 5th ed., 1940. (2) Bonney, V. B., and Rowe, S. C., J. Assoc. Oficial Agr. Chem., 19, 607 (1936). (3) Hassid, W. Z., McCready, R. M., and Rosenfels, R. S., IND.ENG.
CHEM.,ANAL.ED., 12, 142 (1940). (4) Kertesr, Z. I., Food Industries, 6, 168 (1934). ( 5 ) Pucher, G. W., and Vickery, H. B., IND.ENG.CHEM.,h 8, 92 (1936).
. 4 ~ ED., .
OUTSIDE Publication 3691, Bureau of Agricultural Chemistry and Engineering, U. S. Department of Agriculture.
Separation of Carotenes from Xanthophylls A. J . HAAGEN-SRIIT, C. E. P. JEFFREYS, AND J. G . KIRCHNER William G. Kerckhoff Laboratories o f the Biological Sciences, California I n s t i t u t e o f Technology, Pasadena, Calif.
T
HE methods which have been most generally used for the separation of xanthophylls from carotenes in provitamin A analysis suffer from certain disadvantages. The separation by extraction of petroleum ether solutions with 85 t o 90 per cent methanol and the separation on various adsorption columns are time-consuming. The obvious advantages of the chromatographic method for routine work are offset b y the difficulties in obtaining adsorbents of uniform performance, and by the fact that there always appears to be some loss of provitamin A material on the columns. It was known that compounds containing conjugated double bond systems such as occur in the azulenes (6) react u-ith 85 per cent orthophosphoric acid to form blue substances which are extracted from petroleum ether by the acid. It was thought that this reaction might be applied to carotene separations. Petroleum ether solutions of the mixed pigments of canned pineapple, which were obtained by saponification of the solid material with 30 per cent potassium hydroxide in methanol for 12 hours at 3" C. and extraction with petroleum ether (60 to TO"), were treated with phosphoric acid; 25-ml. portions of the petroleum ether solut,ion,which does not need to be dried, were shaken viith 4 to 5 ml. of 85 per cent phosphoric acid in a 25-m1. glassstoppered graduate. The acid layer became blue-green in color. The absorption curve for the pigment remaining in the petroleum ether solution was essentially the same as those obtained with carotene solutions which had been purified by the methanol separation and by passage through magnesia and calcium phosphate columns. Figure 1 shows a group of such curves compared uith that of pure @-carotene(S. 11.A. @-carotene),
The xanthophyll fraction obtained from a sample of these mixed pigments by the methanol separation was transferred into petroleum ether and this solution was treated with 85 per cent phosphoric acid. All the pigment was removed from the petroleum ether phase and converted into blue-green substances in the acid layer. On t'he other hand, carotene extracts, purified both by the methanol separation and by passage through magnesia or calcium phosphate columns, gave no apparent reaction when treated in the same manner with 85 per cent phosphoric acid. The absorption curves of such extracts were not appreciably altered by the phosphoric acid treatment (Figure 2). The deviation of the curve of a sample of phosphoric acid-treated 6-carotene from that of the untreated carotene is similar to that obtained with extracts of natural materials. This change in the absorption of the pigments is undoubtedly due to isomerization of the type first observed by Gillam and El Ridi (3, 4) and extensively studied by Zechmeister and his co-workers ( 5 , 7 , 8 , 9 ) , and by Beadle and Zscheile ( 2 ) . Using this shortened method, results were obtained on canned pineapple which were in satisfactory agreement with those of biological assays (Table I). I n this case the results can be expressed as units of vitamin A, since chromatographic adsorption on magnesia showed the pigment to be homogenous, and the absorption curre checked that of pcarotene which had been treated with phosphoric acid. A comparison was made of the A. 0. A. C. method ( 1 ) and the phosphoric acid method, and since carotene has a slight solubility in 90 per cent methanol the results are as might be expected (Table 11). I n each case the phosphoric acid method gave results which were slightly higher t,han those of the A. 0. A. C. method.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 15, No. 3
TABLEI. COMPARISONOF PHOSPHORIC ACID METHOD AND BIOLOGICAL ASSAYFOR VITAMINA ACTIVITYOF FIVEBRANDS OF CANNEDPINEAPPLE (International units per 100 grams) Brand A B C D E
HaPOr Method 135 106 129 134 135
R a t Assay u. s. P. 145 125 143 138 147
TABLE 11. COMPARISON OF PHOSPHORIC ACIDAND A. 0. A. C. METHODS Sample
Hap04 Method
A. 0. A. C. Method
Y / 1 O O 0.
Y / l O O 0.
75.6 111.5 103.9
66.0 99.5 97.3 58.1 90.0 87.6 83.1
66.0
Av.
99.5 97.3 92.3
FIGURE 2. ABSORPTIONCURVE OF P-CAROTENEBEFORE AND AFTER TREATMENT WITH PHOSPHORIC ACID
- - - Pure j3-oarotene -Pure @-carotene after
shaking with phosphoric acid
TABLE 111. COMPARISON OF PHOSPHORIC ACIDAND A. 0. A. C. METHODSON MIXED POULTRY FEEDS Sample 1 2 3
1
I
400
450
500
rnk
FIGURE 1. COMPARISON OF ABSORPTION CURVES OF CAROTENE OBTAINEDBY VARIOUSSEPARATION METHODSAND OF PURECAROTENE I. Adsorbed on, Micron Brand magnesia 11. Separated w t h phosphoric acid reagent 111. Partition between petroleum ether and 90 per cent methanol IV. Pure @-carotene V. Filtered through dicalcium phosphate column
As a further check on the method, samples of mixed poultry feeds were saponiiied and extracted by the A. 0. A. C.procedure. These extracts were treated with 85 per cent phosphoric acid and the absorption curves of the remaining pigments in the petroleum ether solution were measured. The curves were the same as those obtained for extracts separated by the use of 90 per cent methanol, and the concentrations of carotene calculated from the absorptions at 450 mp were in agreement (Table 111).
Summary A new method which has been applied to the purification of carotene extracts in the determination of carotenes in foods, etc., makes use of the reaction between xanthophylls and 85 per cent orthophosphoric acid to separate the latter from carotene. Results agreed satisfactorily with the biological assays and also with the A. 0. A. C. method. It is
H ~ P O Method P
A. 0. A. C. Method
Y/Q.
Y/%
13.3 15.3 13.8
11.0 12.4 11.5
more rapid than the A. 0. A. C. method and probably more accurate for analysis of material of low or average carotene content. This is due to the loss of carotene in the A. 0. A. C. method occasioned by the residual solubility of carotene in 90 per cent methanol. When the spectrophotometer is used it is preferable to make the measurements in the range of 440 to 450 mp, since the absorption values of the extracts do not deviate greatly from those of @-carotene. As in all analytical methods, this method must be used with discretion, as, for example, phosphoric acid will not remove lycopene. It will not separate a- and &carotene, and in such cases the method would yield the crude carotene content.
Acknowledgment The authors wish to acknowledge with thanks the Pineapple Producers Cooperative Association's financial support for the research program of which this investigation is a part.
Literature Cited (1) Assoc. Official Agr. Chem., Official and Tentative Methods of Analysis, 1940. (2) Beadle, B. W.,and Zscheile, F. P., J . Bid. Chem., 144,21 (1942). (3) Gillam, A. E.,and El Ridi, M. S., Biochem. J.,31, 1605 (1936). (4) Gillam, A.E.,and El Ridi, M. S., Nature, 136,914 (1935). (5) Polgar, A,, and Zechmeister, L., J . Am. Chem. SOC., 64, 1856 (1942). (6) Sherndal, A. E., Ibid., 37, 167 (1915). (7) Zechmeister, L.,and Schroeder, W. A.,Ibid., 64,1173 (1942). (8) Zechmeister, L.,and Tusson, P., Ber., 72, 1340 (1939). (9) Zechmeister, L.,and Tusson, P., Biochem. J., 32,1305 (1938).
