Chemical Determination of Vitamin C. R. THOMPSON', M. A. EWAN,
A
in Dried W h o l e Eggs
S. M. HAUGE, B. B. BOHREN, AND F. W. QUACKENBUSH
Purdue University, Lafayette, Ind.
A chemical method i s described for the determination of vitamin A in dried whole eggs b y the use of chromatographic adsorption. After the sample i s hydrolyzed with alkali and extracted with ethyl ether, the unsaponifiable fraction i s adsorbed on a column of calcium hydroxide and the p-carotene, cryptoxanthol, vitamin A, luteol, and zeaxanthol bands are allowed to separate. The provitamins are eluted and determined separately. Antimony trichloride reagent is then added to the combined vitamin A and provitamin A eluates and the amount of blue color determined in a photoelectric colorimeter. Data presented on nine samples of dried eggs show that the values obtained b y the chemical method agree within 10% with those obtained b y bioassay,
A
TTEMPTS to use published chemical methods ( 5 , 7 ) for the determination of vitamin A in dried eggs in this laboratory failed to give values in agreement with those obtained by bioassay. Investigation showed that differences resulted from poor extractability of the vitamin and errors caused by the presence of large amounts of carotenols for which arbitrary correc, tions were not reliable. When extraction of dried eggs in the Waring Blendor was tried as outlined in the method of Schrenk, Chapin, and Conrad ( 7 ) , a large amount of vitamin A remained in the residue, The residue was hydrolyzed with alkali and extracted with ethyl ether, after which the light absorption of the extract was measured at 326 and 450 millimicrons. The values obtained indicated the presence of substantial amounts of both vitamin A and carotenoids. Values obtained by the Carr-Price reaction further substantiated the presence of vitamin A. Bioassays of the extracted residue provided further proof that ethyl ether extraction of the whole egg does not remove vitamin A quantitatively. In this experiment about 200 grams of a dried egg sample were extracted with ethyl ether in a large Soxhlet extractor for 24 hours. The residue showed a biological potency equivalent to 14.5 I.U. of vitamin A per gram. Various solvents were used in attempts to remove the vitamin A quantitatively. A 45-gram sample of dried egg was extracted with about 250 ml. of solvent in a Soxhlet extractor. To test the completeness o i extraction, a sample of the residue was hydrolyzed with alkali, the unsaponifiable matter was extracted with ethyl ether, and the amount of vitamin A was determined by the CarrPrice reaction. The results (Table I) showed that ethyl ether was the poorest solvent of those tried for the extraction of vitamin A. However, the solvents which were most effective for the extraction had relatively high boiling points, and prolonged heating resulted in darkening of the extract. Large amounts of carotenols in the dried eggs were shown to cause substantial errors when attempts were made to determine the vitamin A by means of the Carr-Price colorimetric procedure as modified by Koehn and Sherman ( 5 ) . Klose, Jones, and Fevold (4) reported satisfactory agreement of results obtained by this method with those shown by bioassay. When this procedure was used with the total unsaponifiable extract the values were consistently higher than those with the bioassay procedure (Table 111, column 3). Attempts to employ an arbitrary factor to correct for carotenoids were unsuccessful. Reliable results were obtained by chromatographic separation of the unsaponifiable extract obtained after direct alkaline hydrolysis of the whole egg. Gillam and Heilbron ( 2 ) had shown that the vitamin A of egg yolk could be separated from xantho~ ~ 1
phylls by adsorption on calcium carbonate and elution with petroleum ether-benzene. However, no attempt at quantitative separation was made. Preliminary trials in this laboratory revealed that calcium hydroxide was a better adsorbent than calcium carbonate for the chromatographic separation of the carotenoids of eggs. When the unsaponifiable extract was adsorbed from a petroleum ether solution and the column developed with a mixture of 60% benzene and 40% petroleum ether a sharp separation was obtained. The carotenoids were eluted from the column in the following order: p-carotene, cryptoxanthol, vitamin A, luteol, and zeaxanthol. The identity of p-carotene, cryptoxanthol, and luteol wm established by comparing light absorption curves (Beckman spectrophotometer, Model DU) with the curves of the pure materials (8). The eeaxanthol band contained large amounts of isomerized material as indicated by comparison with a curve of pure aeaxanthol. The amounts of &carotene, cryptoxanthol, and total carotenoids agreed well with the values obtained by Schrenk et al. ( 7 ) (Table 111). A satisfactory light-absorption curve on the vitamin A eluate was not obtained. Contributing t o this failure was a contaminant dissolved from the calcium hydroxide and Hyflo Super-Cel which absorbed in the region of 310 to 370 millimicrons. The presence of this material was shown by allowing the benzene-petroleum ether solution to run through fresh adsorbent and determining the light absorption of the percolate. This material gave no color with antimony trichloride but attempts to remove it completely before chromatographing were unsuccessful. Preliminary attempts to determine vitamin A concentration by direct spectrophotometric measurements on the eluate were discouraging. This was consistent with the observations of Hauge, Zscheile, Carrick, and Bohren ( 3 ) who found that the curves given by the total carotenoids of fresh eggs as well as dried eggs were very different from that of vitamin A alcohol, both in position of maximum and in shape. Denton, Cabell, Bastron, and Davis (1) found they could not determine vitamin A in dried eggs spectrophotometrically because impurities absorbing in the region of the vitamin A maximum did not remain constant during storage. Adsorption and elution of vitamin A from the calcium hydroxide chromatogram were shown to be satisfactory (Table 11). Crystalline vitamin A alcohol and the unsaponifiable fraction from standard U.S.P. reference cod liver oil were used in these tests. I n each case the vitamin was adsorbed from petroleum ether solution and eluted with benzene-petroleum ether. The solvent was removed from the eluate, the vitamin A redissolved in chloroform and treated with antimony trichloride reagent,
Table
I.
Efficiency of Solvents in Extraction of Vitamin A from Dried Whole Egg" p e r Cent of Initial Vitamin A Extraction Weight of Egg Content of
Solvent E t h y l ether
Ethyl alcohol Absolute alcohol
1 $gg~~~~~~~ealcohol} Carbon tetrachloride
, $
Time Hours 30 89 42 89
Extracted
38.0 41.7 51.0 51.5
9.3 9.7 2.3 2.3
42
50.0 51 .O 43.9 46.5 45.5
2.0 2.3 2.0 1.5 3.7
89
94 94 42
Vitamin -4 content by bioassay, 60 I.U. per gram.
Present addrass, U. 9. Army Medical Nutrition Laboratory, Chicago 9,
Ill.
113
Residue
I. U./oram
INDUSTRIAL AND ENGINEERING CHEMISTRY
114
aiid the light transmission determined with an Evelyn photoelectric colorimeter. Further studies showed that vitamin A could be separated from luteol. This pigment followed vitamin A closely on the chromatogram. METHOD
For the hydrolysis, a 5.0-gram sample is weighed into a 125-ml. Erlenmeyer flask, and 20 ml. of absolute methanol and 5 ml. of saturated aqueous potassium hydroxide are added. The contents are stirred with a glass rod until complete suspension of the sample is effected. The flask is then heated on a steam bath for 10 minutes or uiitil the dried egg particles are disintegrated. The hydrolyzate is then cooled and transferred with 70 ml. of water to a 500-ml. separatory funnel. The first extraction is made with 35 ml. of peroxide-free ethyl ether and the four subsequent extractions with 25- t o 30-ml. portions. The last extractions should be almost colorless. The ether extract is washed five times with 25-m1. portions of water, after which it is dried over 20 grams of anhydrous sodium sulfate for 1 hour a t room temperature. The ether extract is evaporated to approximately 15 ml. under reduced pressure in a 50" C. water bath and then transferred to a 25-ml. volumetric flask with dry petroleum ether. If the solution still contains some moisture, as indicated by cloudiness, it should be dried with a small amount of sodium sulfate. I n the chromatographic separation 10 ml. of this solution are adsorbed on a column (20 X 135 mm.) of 3 parts of calcium hydroxide (Braun's Lot 90. 10,588) and 2 parts of Hyflo Su er Cel. The chromatogram is developed with a mixture of 60% Eekene (thiophene-free) and 407, dry petroleum ether. The two lowest bands containing &carotene and cryptoxanthol are eluted separately. The vitamin A fraction is collected until the luteol begins to give a yellow color t o the eluate. If the column is properly packed, the 6-carotene and cryptoxanthol bands are easily distinguished. The luteol band should be sharp a s i t nears the bottom of the column. Fifty t o 80 I.U. of vitamin A and 150 t o 200 micrograms of total carotenoids (as 6-carotene) can be handled satisfactorily with the column described. A total volume of 250 to 500 ml. of combined 6-carotene, cryptoxanthol, and vitamin A eluate is the optimum range for best results.
Table II.
Recoverv of Vitamin A from a Calcium Hydroxide Chromatogram Vitamin A Vitamin A
Vol. 18, No. 2
After removing the solvent from the 8-carotene and cryptoxanthol fractions, they are taken up in 10 ml. of petroleum ether and the light transmission is measured by means of the Evelyii photoelectric colorimeter with the standard 440 mp filter. Thew solutions are then recombined with the vitamin A fraction and the solution is evaporated under reduced pressure a t 50" C. to approximately 15 ml. This solution is then transferred to a 25ml. volumetric flask with redistilled chloroform. Chloroform is used in this transfer because it is a somewhat better solvent thaii petroleum ether. Ten milliliters of this solution are evaporated to dryness in a colorimeter tube and redissolved in 2 ml. of chloroform. The tube is placed in the E-velyn instrument, 8 ml. of antimony trichloride reagent are added, and the light transmission is measured with the standard 620 filter. The blue color should be read within 5 to 10 seconds after the antimony trichloride reagent is added. This color should be brilliant and clear. Cloudiness in the solution a t this point usually indicates insufficient care in excluding adsorbent or moisture. One gram of U.S.P. rcfercncc cod liver oil No. 2 was saponified and extracted by the above procedure in establishing the calibration curve for the vitamin A . .4 sample of p-carotene, purified by chromatography, was used as the standard for the determination of @-carotene,cryptoxnnthol, and total carot,enoids. RESULTS AND DISCUSSION
Results obtained on dried egg samples with this method were reproducible and showed good agreement with those obtained by bioassay (Table 111). Eight samples of dried eggs of different storage history were tested for vitamin A by both methods with a maximum deviation of 10% in results. Eight replicate chemical determinations on a single sample showed similar reproducibility. Several precautions were found necessary with respect to materials and procedure; other precautions were taken without testing their importance. One of the most important precautions to be observed in using this method is the preparation of the petroleum ether. Some samples of commercial petroleum ether contain an impurity which produces a green color when mixed with the antimony trichloride reagent. The use of such petroleum ether without purification results in erratic vitamin .4 values. The petroleum ether was purified as follows:
Four to five gallons of commercial petroleum ethcr (boiling point 65' to 67" C.) R-ere percolated through a column (7 x 35 cm.) of silica gel (The Davison Chemical Corporation, S o . 659,I.u. I. u. 70 528-2000). The Dercolate was then Dlaced in a 5 - d I o n flask and U.S.P. reference cod liver oil No. 2 24 26 108 &red mechanicaky for several hours with each of two successive 122 115 95 portions of concentrated sulfuric acid. The two phases were 235 243 103 Crystalline vitamin A alcoholo 19 7 19 0 96 separated and the last trace of acid was removed by treatment 20 3 18 5 91 with a dilute solution of sodium hydroxide. The petroleum ether was then stirred for a few hours 0 Purchased from Distillation Products, Inc. with alkaline potassium ucrmanganate, after diich- it was drawn off, distilled, and dried over anhydrous sodium Table 111. Comparison of Chemical and Bioassay Methodsa Chemical Methods sulfntc. Adsorbed
Source of Vitamin A
Sample
3B 4B 5B 8B 9n 10B 12B 13B 14
Not Chromatographed Chromatographed Total caroteVitamin noidsb A0 Carotene xantiolb A A aotivityd
Micrograms/ gram 100.6 106.9 102.0 70.3 114.0 101.3 79.5 72.9 98.2 94.5 95.0 97.6 91.9 97.7
ii.7
I.U . / g r a m 71.4 93.0 75.5 54.7 82.0 74.0 65.0 86:O
..
.. .. I
.
I. L;./gram
Micrograms/gram 3.0 3.2 3.6 3.4 4.3 4.8 2.6 3.0 3.3 33 .. 53 2.9 2.5 2.7 2.2 3.6 2.8c 3.4c
..
.. ..
.. .. ..
,
39.0 31.5 30.7 21.2 3 20 5 .. 2 5 26.0 20.9 35.1 37.0 36.4 36.1 33.9 36.8 6 . 49 33
:&, VitaminA
I. U./gram
46.7 40.2 41.9 28.0 38.5 33.3 32.4 27.6 42.6
44.0 41.0 43.0 30.0 41.0 36.0 32.0 25.0 45.0
43.9 43.6 41.4 44.3 44.4 40.9
..
44.5
I.U. (Chemical) I.U.(Bioassay) X 100
..
..
106 98 98 94 94 93 101 110 95 99 97 97 92 98 99 91
i l l values expressed on moisture-free basis. khprpssed as p-carotene. Calr u1:ited from hlue color given by total uiisnporiifiable extract. ,I 1.U. of vitamin A equivalent t o 0.6 niicrogram of @-caroteneand 1.2 micrograms of cryptoxanthol ( 6 ) . F o u l
