256
INDUSTRIAL AND ENGINEERING CHEMISTRY
Conclusions The data presented show that the electrolytic method described is entirely satisfactory for the determination of iron in tungstic acid and powdered tungsten metal. The method is new in that it offers a means of removing the iron completely from the bulk of the tungsten compound in the form of a tungsten-iron alloy, which on the average contains about 20 per cent of iron. Thus instead of having to make a difficult and perhaps incomplete chemical separation of a few thousandths per cent of iron, it is necessary only to analyze the small amount of alloy thus obtained. Since the iron is finally separated as ferric hydroxide, other ordinary impurities of tungstic acid should not interfere. The results given indicate that iron in tungsten or tungstic acid can bedetermined to within about oeo2 mg* by this electrolytic separation. The iodometric method for the
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quantitative determination of these small amounts of iron was found to be very good and was chosen because it is relatively simple and requires very little experience. However, it is the alloy separation of the iron which is the basis of the method and any other preferred procedure, volumetric or colorimetric, could be used for the final determination of the iron.
Literature Cited (1) Driggs, F. H.,private communication. (2) Fink, C. G., and Jones, F. L., Trans. Electrochem. SOC.,59,461
(1931).
(3) Grey,
E. C.,J. Chem. SOC.,1929,35. (4)Holt, M.L., Trans. Electrochem. Soc., 66,453(1934). (5) Smithell, “Tungsten”, p. 17, New York, D. Van Nostrand Co., .nnn
1Yi)O.
(6) Thompson, M. de Kay, and Rice, C. W., Trans. Electrochem. SOC., 67,71 (1935).
Determination of Carotene in Silage An Improved Method D. M. HEGSTED, J. W. PORTER, AND W. H. PETERSON University of Wisconsin, Madison, Wis.
A
LTHOUGH the old Willstatter-Stoll (I??) method for the determination of carotene has been modified and improved in recent years, all modifications (2, 7, 9) depend upon pigment distribution between petroleum ether and alcohol (90 per cent methyl or 85 per cent ethyl) for the separation of carotene and Xanthophylls. This procedure has not proved reliable when applied to silage. Kane and Wiseman (4)found spectrographic evidence for pigments other than carotene in the petroleum ether solution of A. I. V. (acidtreated) silage. Peterson et al. (6,6)showed that the carotene content of some A. I. V. silages was higher than that of the forage used in their preparation, and suggested that this might be due to pigments developed in the silage. Other workers (3, 22) have also found abnormally high carotene values for A. I. V. silages. Subsequently Quackenbush, Steenbock, and Peterson (8) found that thecarotene fraction obtained from A. I. V. alfalfa silages by the petroleum ether-ethyl alcohol procedure contained three pigments other than carotene. These pigments were separated from carotene by the use of magnesium oxide chromatograms and were found to be biologically inactive. Since these pigments amounted to as much as 40 per cent of the so-called carotene, the figures for such silages obtained by the petroleum ether-alcohol procedure are obviously too high. I n an attempt to find a method, other than the tedious chromatographic procedure which would separate the pigments, several solvents were used in place of 85 per cent ethyl alcohol. As a check on the effectiveness of the solvent, the carotene and noncarotene contents of the fractions were carefully determined by chromatographic separation. Clausen and McCoord ( I ) had used diacetone for the separation of the carotenoid pigments in blood and this solvent proved satisfactory for the separation of those in silage. While absolute separation of pigments with such similar solubilities is probably impossible, nearly quantitative results were obtained.
Procedure A 500- t o 1000-gram sample of fresh silage is well mixed and ground. Twenty-five grams are weighed into a flask and 200 cc. of alcohol are added immediately. The mixture is refluxed for 40 minutes and the alcoholic solution decanted. The residue is extracted again by refluxing 40 minutes with another 200-cc. portion of alcohol and the solution decanted. To the combined alcoholic extracts 40 cc. of 20 per cent alcoholic potassium hydroxide are added, and the solution is shaken and allowed to stand overnight. An alternative procedure to the alcohol extraction is to reflux the sample with alcoholic potassium hydroxide. The determination may then be run immediately. In either case the total volume of the alcoholic extracts must be measured as a basis for the calculation*of the carotene content. To remove any carotene not extracted by the alcohol 80 cc. of Skellysolve Benzine, b. p. 65 to 75 are added to the residue and heated just to the boiling point. The flask is then set aside t o cool. Aliquots equivalent to equal amounts of silage are taken from both the supernatant Skellysolve and the alcoholic extracts. Usually 5 cc. of the Skellysolveextract are placed in a separatory funnel with 2 cc. of 20 per cent alcoholic potassium hydroxide and shaken. Twenty-five cubic centimeters of the alcoholic extract are then added, followed by 15 cc. of Skellysolve and 7 or 8 cc. of water, and the mixture is well shaken. After complete separation of the two layers, the alcoholic solution is drawn off and re-extracted with two more 15-cc. portions of Skellysolve. Three extractions are sufficient to remove all the carotene, although the Skellysolve is still yellow with xanthophylls. The combined Skellysolve extracts in a separatory funnel are washed free of alkali with four 15-cc. portions of water. The noncarotene pigments are then removed by extracting the Skellysolve solution with four 10-cc. portions of a diacetone solution consisting of 100 volumes of diacetone (acetone-free, from Commercial Solvents Corp., Terra Haute, Ind.) and 6 volumes of water. The mixture is shaken vigorously after each addition of diacetone solution, and, after standing until complete separation of the phases, the lower layer is drawn off and discarded. The carotene solution is now washed twice with water to remove diacetone, made to 50-cc. volume, and read upon the spectrophotometer. O,
ANALYTICAL EDITION
MAY 15, 1939
257
procedure in removing the noncarotene pigments, and also to TABLEI. LOSSESIN MAGNESIUM OXIDE CHROMATOGRAM determine the amount of carotene removed by the hypophasic Pigments Separated solvent. The Skellysolve solutions were washed three times ApparNoncarowith water to remove any diacetone, filtered through a little ent tene as Hypophasic CaroCarocamReanhydrous sodium sulfate, and taken to dryness under reSample Solvent tene tene tene covery duced pressure. The pigments were then taken up in a M d l c g . Me./kg. Me./ko. % small amount of Skellysolve and forced through the chromatoGrass silage 85% EtOH 718 338 77 58 36 78 Diacetone 678 493 gram with pressure. The columns were developed with A. I. V. alfalfasilage 85% EtOH 215 117 36 85 11 84 Diacetone 168 132 Skellysolve, followed by Skellysolve containing small amounts Molasses alfalfa 85% EtOH 181 118 55 95 of absolute alcohol as described by Quackenbush et al. (8). 138 109 3 81 silage Diacetone 10 85 Green alfalfa 85% EtOH 201 161 The pigments in the hypophasic solution were first transferred 196 160 5 84 Diacetone to Skellysolve by dilution of the solution with water and extraction with Skellysolve, and then treated in a similar manner. The fractions collected from the column were diluted to Comparison with Other Methods proper volume with Skellysolve and read in the spectroThis procedure and a similar one using 85 per cent ethyl photometer. Both the carotene and noncarotene pigments alcohol or 90 per cent methyl alcohol to remove the hypo1 Yo are reported in terms of carotene based upon E 480 = 2150 phasic pigments were compared upon numerous samples. 1 cm. To eliminate sampling errors equal aliquots of the original which was determined with pure P-carotene. extracts from one sample of forage were taken. The value obtained by reading the final Skellysolve solution in the spectrophotometer has been designated “apparent carotene” Results and Discussion in the tables, since this solution may contain considerable Since Quackenbush et al. (8) had shown that magnesium amounts of other pigments. If a quantitative separation is oxide columns give satisfactory separation of the pigments, obtained, this is the actual carotene content. this adsorbent was tried. Recovery from these columns, Both the hypophasic and epiphasic solutions were subjected however, was neither quantitative nor consistent. I n Table I to chromatographic analysis to determine the efficiency of the
--
d
TABLE 11. Sample
Forage
NO.
COMPARISON OF DIACETONE AND ALCOHOL METRODR Hypophasic Solvent
Apparent Carotene
Mo./ko.
(1
1
A. I. V. alfalfa silage
2
A. I. V. alfalfa silage
3
A. I.
V. alfalfa silage
4
A. I.
V. alfalfa silage
5
A. I. V. alfalfa silage
6
A. I. V. grass silage
7
A. I. V. pes. silage
8
Phosphoric acid alfalfa silage
9
Phosphoria acid alfalfa silage
10
Molasses alfalfa silage
11
Molasses alfalfa silage
12
Molasses alfalfa silage
13
Molasses alfalfa silage
14
Corn and soybean silage
15
Corn silage
16
Corn silage
17
Untreated alfalfa silage
18
Green corn
Extracted with five 10-cc. portions of diacetone.
85% EtOH Diacetone 85% EtOH Diacetone 85% EtOH Diacetone 85% EtOH Diacetone 85% EtOH Diacetonea 85% EtOH Diacetonea 85 EtOH 90% MeOH Diacetone 85% EtOH Diacetone 85 EtOH 90% MeOH Diacetone 85% EtOH Diaoetone 85% EtOH Diacetone 85% EtOH Diacetone 850/ EtOH 90% MeOH Diacetone 85% EtOH Diacetone 85% EtOH Diacetone 85% EtOH Diacetone 85 EtOH 90% MeOE Diacetone 85% EtOH Diacetone
285 252 264 248 332 281 322 310 181 156 435 382 143 149 120 238 208 246 276 217 140 130 505 460 244 202 171 171 156 64 52 189 167 59 62 68 61 46 256 243
--Pigments Carotene Me./kg. 195 224 176 192 250 244 248 265 151 151 371 364 103 103 109 197 197 205 205 205 114 119 442 352 206 211 144 144 144 47 47 134 134 40 47 47 47 47 238 238
Separated--.
Non-
aarotene Me./ke. 45 22 64 24 70 29 45 17 33 5 52 12 34 34 11 46 12 36 41 5 21 10 45 18 33 5 20 20 4 6 0 32 16 10 4 11 11 0
12 0
Recovery
% 84 98 91 87 96 97 91 88 102 100 97 98 96 92 100 102 100 98 89 97 96 99 96
so
98 107 96 96 95 83 90 88 90 85 98 98 95 107 98 98
Carotene in Hy ophaaic Sorvent Mo./ke.
..6 ..
24
..
12
.. .. 3
16
3 24
.... ..
.. .. .. .. .. 5 ..9
12
*.
10
..
.. ..
..
..
..
8
..
..
.. .. ..
..
INDUSTRIAL AND ENGINEERING CHEMISTRY
258
are shown a few typical results in which the recovery varied from 58 to 95 per cent. Similar losses with aluminum oxide columns have been reported by Willstaedt and With ( l a ) . The table does show, however, that the apparent carotene was higher when alcohol was used to remove the hypophasic pigments, and indicates that this difference was due to the higher content of noncarotene pigments in the solution. Magnesium oxide was discarded and several new adsorbents were tried. Calcium carbonate proved satisfactory. The carotene could be washed out of the column with Skellysolve and then the noncarotene pigments could be removed with Skellysolve containing 10 per cent of absolute alcohol. The results obtained with numerous samples are shown in Table 11. The column headed “apparent carotene” represents, as before, the reading of the Skellysolve solution before chromatographic analysis. The next three columns give the actual carotene and noncarotene pigments separated from this solution and the percentage recovery from the calcium carbonate chromatogram. Column 8, “carotene in hypophasic solvent”, gives the carotene found in the diacetone or alcoholic solution when subjected to chromatographic analysis. While there are occasional losses during the manipulations, the results clearly indicate that the apparent carotene values obtained from silages when either 85 per cent ethyl or 90 per cent methyl alcohol is used are considerably too high. This error is the result of the pigments other than carotene which remain in the Skellysolve solution. When diacetone is used, the error is greatly reduced, the apparent carotene nearly equaling the actual carotene. The loss of carotene in the diacetone solution is no disadvantage and actually decreases the error of the determination. Since some noncarotene pigment remains in the carotene fraction, and an almost equal quantity of carotene is removed, these errors cancel each other. When this is considered, the apparent carotene is practically equal to the actual carotene content. A calculation of the errors illustrates this: I n sample 8 the apparent carotene value obtained with alcohol is 238 and the actual carotene content 197. If we assume that about 3 mg. were extracted by the alcohol, the total carotene content was 200 38 and the percentage error = X 100 = 19 per cent. In the case of diacetone the total carotene was 197 12 = 209, which is equal to the apparent carotene figure. Some of the inconsistencies in the data are due not only to losses during the manipulations, but also to the errors involved in reading the spectrophotometer. These have been discussed by Shrewsbury et al. (IO) and in the authors’ experience appear to be about *3 per cent. Since readings were necessary upon three or four fractions of each sample, the combined errors may in some cases account for high or low recoveries, and also for the difference between the total carotene obtained from one sample by the two methods. A further advantage of the diacetone method is that four or five washings are sufficient to remove the interfering pigments. The results with alcohol may be improved by numerous washings, but the amount of pigment removed each time is very small and it is impossible to tell when the removal is complete. Carotene is also lost in the alcohol, and thus the results may be either high or low, depending upon the number of washings. The recovery of known amounts of carotene which were added to the crude alcohol extracts is shown in Table 111. These values are the apparent carotene values after washing with diacetone and show satisfactory recovery of the added carotene. Quackenbush et al. (8) have shown that at least some of the interfering pigments are produced by the action of acid upon lutein. I n Table IV are shown the results obtained by the
wo
+
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alcohol and diacetone methods upon a sample of phosphoric acid alfalfa silage which was analyzed before and after treatment with acid. Two 25-gram samples were taken and placed in alcohol. To one were added 2.7 cc. of a 2 N mixture of hydrochloric and sulfuric acids. After standing overnight the two samples were extracted as usual and analyzed by both procedures. The use of 85 per cent alcohol gave much larger errors after acid treatment, whereas the error was only about 3 per cent in both samples when diacetone was used.
TABLE111. RECOVERY OF ADDED CAROTENE Carotene Carotene Carotene Added Found Calculated Micrograms Micrograms Micrograms A. I. V. alfalfa silage 0 139 93 237 232 A. I. V. alfalfa silage 0 109 00.5 177 170 Molasses alfalfa silage 0 60.5 137: 137 Corn silage 0 21 60.5 79 81 Sample
Accuracy
% 102 104 98.5 97.7
TABLEIV. EFFECTOF TREATING SILAGE WITH ACID Sample
Hypophasic Solvent
Phoaphorio acid silage AnalyFed before 85% EtOH adding acid Diacetone Analyzed after treatment with 85% EtOH Diacetone acid
Pigments Separated NOUcarotene Mo./ke.
Apparent Carotene Mg./kg.
Carotene Mo./?w.
198 174
104 104
29 5
290 174
159 109
120 5
Summary An improved method for determining the carotene content of silages is reported. The improvement consists of the use of a diacetone solution (100 volumes of diacetone, 6 volumes of water) in place of the usual 90 per cent methyl or 85 per cent ethyl alcohol for removal of the pigments other than carotene. The greater accuracy of the method was shown by the determination of the actual carotene contents by chromatographic technique.
Acknowledgment The authors are indebted to I?. W. Quackenbush for suggesting the use of diacetone and for other suggestions in working out the method.
Literature Cited Clausen and McCoord, J.Biol. Chem., 113, 89 (1936). Guilbert, IND. ENQ.CHEM.,Anal. Ed., 6,452 (1934). Hayden, Perkins, Krauss, Monroe, and Washburn, Ohio Agr. Expt. Sta., Bimonthly Bull. 22,21 (1937). Kane and Wiseman, J. Dairy Sci., 19,447(1936). Peterson, Bird, and Beeson, Ibid., 20, 611 (1937). Peterson, Bohstedt, Bird, and Beeson, Ibid., 18, 63 (1935). Peterson, Hughes, and Freeman, IND. ENG.CHEM.,Anal. Ed., 9,71 (1937). Quackenbush, Steenbock, and Peterson, J. Am. Chem. SOC., 60, 2937 (1938).
Schertz, Plant PhysioZ., 3, 211 (1928). Shrewsbury, Kraybill, and Withrow, IND.ENQ. CHEM.,Anal. Ed., 10, 253 (1938). Virtanen, Biochem. 2..258, 251 (1933). Willstaedt and With, 2.physiol. Chem., 253,40 (1938). Willstitter and Stoll, “Untersuchungen iiber Chlorophyll. Methoden und Ergebnisse”, Berlin, Julius Springer, 1913. PUBLISHED with the approval of the Direator of the Wisconsin Agricultural Experiment Station.
