107
V O L U M E 1 9 , N O . 2, F E B R U A R Y 1 9 4 7 T a b l e 111. :ample 1A 2A
1B 2B
Aloe-Emodin in Aloes
Weight Gram 0,5000
Colorimetric
0.5000
0.043
0.5000 0,5000
70 0.045
...
...
Color Poldroaraphic
.
%
... 0:036
0.042
lecting the washings in the flask, and fill the flask t o the mark with chloroform. If the solution is yellow, measure the extinction a t 450 millimicrons; otherwise use a wave length in the region 490 to 530 millimicrons. Calculate the amount present from a calibration curve (usually a straight line) prepared using a solution containing 0.500 gram of aloe-emodin per liter of chloroform and dilutions of the concentrated solution as necessary. Portions of the aloes samples used for the aloin determinations listed in Table I were analyzed for aloe-emodin With these particular samples the chloroform extract Tvas yellow and so a wave length of 450 millimicrons was used. The results are given in Table 111, which includes some polarographic results tor comparison.
It is also possible t o extract the aloe-emodin from the chloroform solution with 0.1 N sodium hydroxide. Since the aloeemodin is an anthraquinone, i t can be determined by polarographic methods, but in this particular case the polarographic method is not recommended because of the extra steps involved. ACKNOWLEDGMENT
The authors wish to thank the Wallace Laboratories for their generous gift of materials and funds that made this investigation possible. LlTERATURE CITED (1) Cahn. R. S.. and Simonsen. J. L.. J . Chem. SOC..1932. 2573. (25 Furman, N: H., and Bricker, C. E., and Whitesell, E. B., IND.
ENG.CHEM., ANAL.En.. 14, 333 (1942). (3) Gardner, J. H., and Joseph, W., J . Am. Pharm. Assoc., 26, 794 (1937). (4) Tutin, F., and Naunton, W. J. S., Pharm. J . , 91, 836 (1913). (5) Viehoever, A., Am. J . Phurm., 107, 47 (1935). ABSTRACTEDfrom the thesis submitted by K. G . Stone in partial fulfillment of the requirements for the degree of doctor of philosophy to the chemistry department, Princeton University, M a y 1946.
Determination of Vitamin A in Fish Liver Oils with Activated Glycerol Dichlorohydrin Comparison with Spectrophotometric and Antimony Trichloride Methods ALBERT E. SOBEL AND HAROLD WERBIN Pediatric Research Laboratory, Jewish Hospital of Brooklyn, Brooklyn, ,V. Y . i c t i i a t e d g l j cero1 dichlorohydrin, a n e w colorimetric reagent for vitamin A, has beeii applied to the determination o f bitamin A on the unsaponifiable fraction o f fish l i \ e r oils. The results were compared to t h o s e obtained b y the a n t i m o n y trichloride a n d ultraviolet absorption m e t h o d s on the same fractions. S o m e determinatiotis were m a d e on whole oils. Since agreement between activated gly cero1 dichlorohydrin a n d a n t i m o n y trichloride m e t h o d s i s close, the authors proposeactibated glycerol dichlorohydrinfor e s t i m a t i n g vitamin i i n f i s h l i v e r o i l s .
R
ECENTLY the authors reported a new colorimetric reaction of vitamin .I which' takes place on the additionof practical (8) or activated ( 7 ) glycerol dichlorohydrin to a solution of vitamin A in chloroform. This new reaction appears suitable for quantitative purposes, as it obeys Beer's law over a reasonable range and offers the following advantages over the widely used Carr-Price reaction: (1) The violet color produced is stable from 2 to 10 minutes after the addition of the reagent, (2) the reagent. is not affected by traces of moisture that occur on the most humid days, (3) no film of antimony oxychloride is left on-the cuvettes, (4)the reagent is practically noncorrosive, and (5) i t is possible to use a photoelectric spectrophotometer (Beckman) in measuring the absorption of the violet color. The purpose of the present investigation was to determine whether activated glycerol dichlorohydrin could be used as a colorimetric reagent for the quantitative estimation of vitamin .?in , fish liver oils. I n order t o evaluate the new method, the results obtained xvere compared to those obtained by the Carr-Price reaction and the ultraviolet absorption method on the same oils. RIost of the determinations were made on the unsaponifiable fraction of the fish liver oils because i t has been shown (3,5 ) that
less accurate results are found when the whole oil is used, particularly on low potency oils. Some whole oil determinatioas were made in the hope that those substances in whole oils which interfere in the other two methods might not interfere when activated glycerol dichlorohydrin is employed. INSTRUMENTS For ultraviolet absorption measurements, the Beckman quartz photoelectric spectrophotometer mas used with a tungsten lamp as a light source. The absorption path of the Corex cells was 1.0 cm. By varying the slit width 100% transmission of the blank was obtained. The sensitivity knob was kept a t three counterclockwise turns from its extreme clockwise position throughout all the measurements. For the glycerol dichlorohydrin and Carr-Price reactions the Coleman universal spectrophotometer, model 11, was employed with absorption cells whose path was 1.3 cm. Per cent transmis sion readings were made on the galvanometer scale after the blank o Photoelectric colorimeters had been set a t 1 0 0 ~ transmission. employing a 555 mp filter may also be used. REAGENTS Antimony trichloride, reagent grade, Merck. Chloroform, analytical reagent, dried over sodium sulfate, dietilled, and kept over the same drying agent.
108
ANALYTICAL CHEMISTRY Table I.
Oil Sample Standard Standard No. 1 Concentrate
Effect of Chloroform Evaporation on E::&. Method) Values of Fish Liver Oil6
Unsaponifiable Unsaponifiable Whole Unsaponifiable
Taken Up in Absolute Alcohol Directly ,1% .1% E 3 0 0 E350 lcm. lcm. E328 E328 .. 100.7 0.64 0.50 ... 100.8 0 . 6 6 0 . 5 3 ... 23.41 0 . 6 6 0 . 6 0 975 ..., , ..
.
.
and
Liz,(SbCla
Taken O p in Chloroform, Evaporated t o Dryness Residue Taken Up in Absolute Alcohol L1% El% E300 E350 lorn. lorn. E328 E328 99.1 0.64 0.50 ... 1 0 0 . 2 0 . 6 4 0 . 5 2 ... 2 3 . 6 5 0 . 6 7 0 60 976 ,, , ,. .,
--
...
Alcohol, 95% U.S.P. grade, distilled over potassium hydroxide pellets. Alcohol, absolute, prepared by distilling commercial absolute alcohol over sodium. Ether, absolute, reagent grade, was distilled and the first and last quarters were discarded (3). The ether was distilled on the same day it was to be used and was immediately tested for peroxides before used. Upon evaporating the ether and adding absolute alcoho1 to the residue, no absorption in the ultraviolet was observed. Glycerol dichlorohydrin, practical grade from Eastman Kodak Company and from the Ohio Chemical Mfg. Company, which call it 1,3-dichloro-2-hydroxypropane. Carr-Price reagent, prepared by dissolving 90 grams of antimony trichloride, from an unopened bottle, in 300 ml. of chloroform a t room temperature. The solution was filtered before use. Activated Glycerol Dichlorohydrin. Antimony chloride, 2% by weight, dissolved in chloroform was added to 1000 ml. of practical glycerol dichlorohydrin. The mixture was vacuum-distilled a t 86" to 92" C. a t 30 to 40 mm. of mercury after the chloroform fraction had been discarded. The reagent prepared in this manner should give an Li::. value (550 mp) (7,8) of from 1150 to 1250 in the Colemen instrument and should be colorless. The reagent was stored a t room temperature in a glass-stoppered bottle. The reagent prepared in this manner was found to be stable (as shown by constant L values with solutions of known vitamin A content, 7 ) for a t least 2 months. The activated reagent may be obtained from the Shohan Laboratories, Kewark 5, X, J. Standard Vitamin A Solution. About 40 to 50 mg. of a vitamin A concentrate, which comes in gelatin capsules, Control KO. PC-3 from Distillation Products, Inc., were diluted in chloroform to give a concentration of vitamin A (as the alcohol) from 2 to 5 micrograms per ml. of solution. The standard solution was not used for more than two consecutive days. The concentrate has an E::&, value in absolute ethanol of 100.75. This value was supplied by the manufacturers and agreed with the value found by the authors. Multiplying this value by 2000 the standard commercial conversion factor ( 2 ) ,the vitamin A potency may be estimated to be 201,500 U.S.P. units per gram. If the extinction of the concentrate is divided by the extinction of crystalline vitamin A alcohol in ethanol, 1780, the vitamin A content of the concentrate becomes by simple proportion 5:65%. SAPONIFICATION PROCEDURE
The procedure described by Oser et al. (3)was followed. It was tested by determining the vitamin A contents of the whole oil used as standard and its unsaponifiable fraction. The vitamin A values found agreed within o.7yOof one another. CARR-PRICE REACTION
One milliliter of chloroform containing between 2 and 5 micrograms of vitamin A was pipetted into a cuvette and 3.0 ml. of antimony trichloride reagent kept a t 25 a C. were added with a fast delivery pipet (Mohr pipet with a wide tip). The maximum absorption was then read on the Coleman a t 615 mp within 5 seconds. The Carr-Price reaction was run on standard solutions containing from 2 to 5 micrograms of vitamin A. Densities were calculated from the observed galvanometer deflections. A graph was constructed of density readings versus concentration. Each new batch of antimony trichloride was checked against standard solutions. If the values did not agree well with those on the previous graph, a new graph was constructed. GDH REACTION
To 4.0 ml. of activated glycerol dichlorohydrin in a 10-ml. glsss-stoppered graduate was added 1.0 ml. of chloroform con-
.
taining between 2 and 5 micrograms of vitamin A. The solution was mixed and placed in a 25" C. water bath for about 1.5 minutes. I t was then poured into a cuvette and its absorption a t 550 mk was read 2 minutes from the time of the addition of the vitamin A. This reaction was run on standard solutions containing between 2 and 5 micrograms of vitamin A, and a graph of density readings versus concentration was constructed for each new batch of activated glycerol dichlorohydrin.
ULTRAVIOLET ABSORPTION MEASUREMENTS
Density readings a t 300,325,328, and 350 mp were made on the Beckman spectrophotometer, using absolute alcohol as the solvent for the whole or unsaponifiable fraction of the fish liver oil. The extinctions of the whole oils were calculated from the observed densities a t 328 mp, while the extinctions of the unsaponifiable extracts were calculated from the observed densities a t 325 mp. The density reading a t 325 mp rather than 328 mp was chosen to calculate the extinctions of the unsaponjfiable fractions of the fish liver oils because the maximum absorption of vitamin -4 alcohol occurs a t 325 mp while the maximum absorption of vitamin 9 esters takes place a t 328 mp. This was also the case with the standard vitamin A concentrate, which gave almost identical extinctions (within 0.770) only when the densities a t 325 mp for the unsaponifiable fraction and at 328 mp for the whole oil iTere used. Although some laboratories use the E;:&. (328 mp) for both the whole and unsaponifiable fractions of fish liver oils ( 2 ) ,the use of E:?&, (325 mp) for the unsaponifiable fraction has been suggested by Gridgeman (1) and Wilkie (11). The whole or unsaponifiable extract of the oil vias diluted so that the densities read between 0.4 to 0.8. Vandenbelt, Forsyth, and Garrett ( I O ) found the best densities for working with vitamin A are from 0.5 to 1.9. This compares with 0.4343, the optimum denPity calculated by Twyman and ,411sopp (9) for use with photoelectric instruments. Rawlings and Wait (6) working with vitamin A on the Beckman model DU spectrophotometer obtained the best reproducibility by working between 0.3 and 0.8 density unit. The vitamin 9 content of the fish liver oil in micrograms was found by dividing the calculated E:?,, by 1780, t h e extinction vitamin A4alcohol, and multiplying the result by lo6. The nurnber of U.S.P. units per gram of oil was Calculated by multiplying E::&, (325) by 2000 for the unsaponifiable extract, andE:?&, (328) by 2000 for the whole oil. Density readings were taken a t 300 and 350 mp to calculate extinction ratios, a term introduced and defined by Oser et al. ( 4 ) as the E:?:, 300/E:,%m,328 and E;?& 350/E:?$. 328. They have shown that crystalline vitamin A has an extinction ratio a t 300 mp of 0.52. This value rises steadily with increased oxidation of the vitamin A alcohol, much more so than the extinction ratio a t 350 mp. Thus, determination of t,he extinction ratio, particularly a t 300 mp, of a given fish liver oil undergoing oxidation gives an indication of the quality of the oil. VITAMIN A DETERMINATIONS ON WHOLE FISH LIVER OILS
From 30 to 200 mg. of whole oil were weighed into a 25.0-ml. volumetric flask and taken up in chloroform. This solution was then diluted t o contain between 2 and 5 micrograms of vitamin A. All dilutions were made a t 25" C. Both the Carr-Price and glycerol dichlorohydrin reactions were run on this diluted solution and on both the standard and recovery solutions. The latter was prepared by adding 5.0 ml. of the standard to 4.0 ml. of the diluted fish oil solution and diluting to 10.0 ml., so that the recovery solution contained from 2 to 5 micrograms of vitamin A per ml. of solution. From the observed per cent transmission, densities were calculated and the vitamin A concentrations in micrograms were
109
V O L U M E 19, NO. 2, F E B R U A R Y 1 9 4 7 Table 11. Determination of Known Amounts of Vitamin A Added to Fish Oils SbCls Method CalcuDiflatedo Foundb ferencec Y Y 70 1.40 2.86 2 90 1.03 2.91 2.94 0.39 2.59 2.60 3.52 3.12 3.23 - 0.00 2.20 2.20 - 3.75 2 40 2 31 117.50 2 17 2.55 - 0.36 2 74 2.73 - 0.74 2 72 2.70 - 1.36 2 94 2.90 3,OO 0.00 3.00 0.29 3 42 4.43 2.26 3.09 3.16 - 0.90 3.33 3.30 2.61 3.07 3 15 3.08 4.22 2.21 3 95 3 85 - 2.53 - 2.12 3.30 3.23 3.41 3.75 9.98 3.23 0.62 3.25 3.21 1.24 3.25 2.98 2.01 3.04 2.98 2.96 f 0.67 3.20 0.00 3.20 3.13 3.06 - 2.23 3.16 3.10 - 1.90 2.93 - 2.73 2.85 Oct. 22,1945 3.31 3.60 8.76 3.42 7.60 3.68 4.01 2.24 4.10 3 41 3.80 +11.42 3.85 4.13 7.28 2.72 2.73 0.37 2.90 2.90 0.00 April 1, 1948 2.95 2.84 - 3.73 3.12 3.04 - 2.56 2.97 2.92 - 1.68 2.80 2.68 - 4.29 2.i5 2.82 2.54 2.95 2.88 - 2.37 2.60 2.54 2.31 2.56 2.60 1.56 3.43 3.38 - 1.45 2.65 2.65 0.00 2.95 3.05 3 04 3.30 3 38 2.42 2.75 2.72 - 1.09 3.17 3.20 0.95 3.74 3.73 - 0.27 3.91 3.75 - 4.10 3 06 2.98 - 2.61 3.i5 3.63 - 3.20 3.16 3.18 0.63 3.47 3.43 - 1.15 3.21 3.25 1.25 3.88 3.98 2.58 4.01 3.98 - 0.75 3.95 3.88 - 1.77 4.02 4.10 1.98 3.63 3.63 0.00 3.66 3.56 - 2.18 3.20 3.20 0.00 3.32 3.36 1.19 3.58 '3.60 0.56 3 56 3.68 3.37
Oil Sample
1. Nonpercomorphum
+ +
Whole
++
Unsaponifiable
2. Cod liver oil
Whole Unsaponifiable
3. High shark
Whole Unsaponifiable
4. Halibut
+ +
Whole
+ +
Unsaponifiable
5. High D
Whole
H. Argentine
Unsaponifiable Whole
shark Oleum perconiorphum Vitamin 4 in sesame oil Oil A
+ ++ +
t-nsaponifiable Unsaponifiable Unsaponifiable
Oil R
Whole
Oil C
Unsaponifiable Whole
++ + + ++
Unsauonifiable
Oil B
Whole Unsaponifiable
Oil C
Whole Unsaponifiable
Oil D
+
-
Whole
+
Unsaponifiable Oil E
++ +
Whole Unsaponifiable
Oil F
Whole rnsaponifiable
Dispersiond Dispersiond Dispersiond Dispersiond Dispersiond
Dispersiond 6 Dispersiond 7
Unsaponifiable L'nsaponifiable Vnsaponifiable Unsaponifiable Unsaponifiable Unsaponifiable Unsaponifiable Unsaponifiable
Dispersiond 8
1-nsaponifiable
Dispersiond 9
L-nsaponifiable
Arerage
s
1 2 3
4 5
error,
+ + +
+
+ ++ ++ 0.94 0.65
Whole Unsaponifiable
G D H Method Calculatedo Y
Foundb
+
CI /U
I
2.85 2.90 2.67 3.18 2.52 2.66 2.21 3.05 2.57 2.87 3.05 3.48 3.08 3.34 3.12 3.19 4.06 3.45 3.48 2.99 2.99 3.02 3.00 3.22 3.17 3.32 3.19
2.78 2.94 2.67 3.18 2.91 2.78 2.50 3.05 2.53 2.93 2.89 3 48 3.10 3.33 3.08 3.30 4 10 3.48 3.63 3 15 3.23 3 02 2.92 3.22 3.08 3.30 3.30
3.10 3.25 4.35 3.42 3.46 2.69 2.80
3.04 3.13 4.35 3.30 3.30 2.75 2.88
++ 2.22 2.86
2.58 3.33
2.50 3.24
- 2.70
2.41 3.07 3.02 3 06 2.65 3.52 2.70 2.88 3.28 2.68 3.02 3.78 3.99 3.19 3.82 3.20 3.46 3.37 3.76 4.00 4.05 4.02 3.32 3.74 3.32 3.33 3.65 3.65
2.44 3.00 2.98 3.06 2.65 3.55 2.68 2.90 3.38 2.65 3.03 3.83 3.98 3.18 3.98 3.20 3.33 3.43 3.77 4.03 4.10
4.08 3.37 3.78 3.35 3.33 3.78 3.78
%-hole * 3.11 Unsaponifiable + 2.11 a -4mount found in original sample amount added (approximately equal quantities). b Amount found in recovery estimation. (Found - calculated) X 100. calculated d Aqueous dispersion of vitamins including vitamin A . Mean error, 70
Differencec
+ 2.45 1.38 -
- 0.00 - 0 00
t15.50 4.50 +13.20
+
0.00
- 1.56
+
2.09
- 5 25 0.00
+- 0.65 0.30 1.28 +- 3.45 ++ 0.98 0.87 + 4.31 ++ 5.35 8.03
!.OO
- +.67 0.00
- 2.83 - 0.60
+
3.45
- 1.93 - 3.70
+
1.18
- 3.50 - 4.62
-
+ -
-
+
3.10
1.24 2.28 1.32 0.00 0.00
0.85
- 0.74
+- 0.70, 3.05 - 1.12
+
0.33
+- 0.25 1.32 - 0.31
+ 4.19 0.00
- 3.76
+ 0.78 + 0.27 + 0.75 + 1,23 4 1.49
+ +
1 51 1.07
+ 0.91 0.00 ++ 3.56 3.56
++ 0.67 1.01 +
*
2.80 1.88
obtained from the previously constructed graphs. The number of U.S.P. units per gram of oil was calculated by multiplying the vitamin A content in micrograms by 3.57. This factor was found by dividing the vitamin A content of the standard control capsule, 201,500 U.S.P. units, by 56,500, the vitamin A content of the capsule in micrograms. For the ultraviolet absorption measurements, the whole oil was weighed into a 25.0-ml. volumetric flask and taken up in absolute alcohol. Readings were taken a t 300, 328, and 350 mp. The vitamin A content was calculated as described under u l t r a violet absorption measurements. VITAMIK A DETERMINATION ON UN SAPONIFIAB LE FRACTIONS OF FISH LIVER OILS
All these determinations were run within one week of the whole oil determinations, so that any differences in the values between whole and unsaponifiable fraction could not be a b tributed to deterioration. The unsaponifiable extract was dissolved in chloroform. The glycerol dichlorohydrin reaction and CarrPrice reaction along with standards and recoveries were run simultaneously and the vitamin A was determined as described for the whole oil. For the ultraviolet measurements from 1.0 to 3.0 ml. of the diluted chloroform solution containing the unsaponifiable extract of the fish liver oil was placed in a 25-m1. volumetric flask and evaporated a t room temperature with a stream of nitrogen. When all the chloroform was evaporated, the residue was dissolved in absolute alcohol, its ultraviolet absorption a t 300, 325, 328, and 350 mp mas measured, and the vitamin A content was calculated as described. That the chloroform evaporation under the conditions described does not destroy the vitamin A can be seen in Table I. The extinction of a whole fish liver oil dissolved in absolute alcohol was 23.41. Another sample of the oil dissolved in chloroform, followed by evaporation under nitrogen of 2.0 ml. of the chloroform, and solution of the residue in absolute alcohol, gave an E:?&, of 23.65. Two samples of the standard control No.. PC-3 were saponified. The unsaponifiable fraction of the first sample dissolved in absolute alcohol gave an E;?;, of 100.8, while the unsaponifiable fraction of the second sample dissolved in chloroform, followed by evaporation of 3.0 ml. of the chloroform solution and solution of the residue in absolute alcohol, gave an E:?&, of 100.2. Further evidence that the vitamin A was not oxidized under the conditions employed is seen from the same extinction ratios found after chloroform evaporation.
'
ANALYTICAL CHEMISTRY
110 Tabel 111. Comparison of Vitamin A Values on Fish Liver Oils %1. SbCla Method
Oil Sample
GDH Method
1cm. X
2000
Method
Extinction Ratio
E300 E350 E328 E328
U.S.P. 4 a .
U.'.S.P. units per gram
1. Nonpercomorphum
Whole Unsaponifiable
2.
Whole
Cod
Unsaponifiable
3. High shark
Whole Cnsaponifiable
4. Halibut
Whole Unsaponifiable
5. High D
Whole
6.
Unsaponifiable Whole
Argentine shark Oleum percomorphum Vitamin A in sesame oil Oil A
Unsaponifiable
F.R.L."
Unsaponifiable
Unsaponifiable Cnsaponifiable
45,700 44,900 44,200 42,500 995 1,040 1,430 1,440 138,000 134,000 124,400 122,800 61,500 60,000 60,200 59,400 8,750 8,780 9,170 144,100 143,700 56,860 56,860 33,150 31,140 177,300 166,900 187,000
45,300 44,600 47,600 43,800 1,300 1,330 1,490 1,580 122,500 126,200 132,300 126,700 60,900 60,500 63,500 64,000 10,440 9,550 9,570 125,600 126,200 58,980 58,980 33,600 32,250 193,700 196,000
...
F.R.L.0 Multiple Level Bioassay
...
46,920 46,740 46,220 46,560 1,830 1,830 1,600 1,620 130,600 129,400 128,600 130,300 62,500 62,400 62,400 62,000 10,960 10,930 10,180 146,680 147,900 61,520 61,520 33,120 32,000 197,440 197,640 200,000
0.66 0.66 0.68 0.68 0.85 0.85 0.71 0.69 0.69 0.68 0.71 0.71 0.63 0.63 0.63 0.63 0.71 0.72 0.67 0.76 0.76 0.68 0.68 0.68 0.69 0.62 0.63 0.60
0.56 0.56 0.55
... ...
3,940 3,935 3,245 3,305
1.28 1.28 1.17 1.18
0.51 0.52 0.46 0.47
... ...
0.60 0.60 0.54 0.53 0.60 0.60 0.57 0.55 0.56 0.55 0.49 0.49 0.62 0.63 0.57 0.57 0.63 0.63 0.57 0.55 0.55
.. ,.
.. ..
... ... ...
...
...
... ... ... ...
... . .
. .
... ...
... , . .
... ...
...
...
Oct. 22, 1945 Oil B
Whole Unsaponifiable Cnsaponifiable Whole
F.R.L.0 Oil C
Vnsaponifiable F.R.L.0
Unsaponifiable
2,295 2,320 1,890 1,970 1,930 2,260 2,120 1,830 1,810 1,950
1,545 1,500 2,015 2,025
...
...
1,310 1,330 1,850 1,905
3,820 3,830 2,995 3,165
...
...
..
..
1.30 1.32 1.30 1.21
0.51 0.51 0.48 0.46
..
..
April 1, 1946 Oil B
Whole Unsaponifiable
Oil C
Whole Unsaponifiable
Oil D
Whole Unsaponifiable
F.R.L." Oil E
Unsaponifiable Whole rnsaponifiable
F.R.L.0 Oil F
Unsaponifiable Whole Vnsaponifiable
F.R.L.5 Dispersion Dispersion Dispersion Dispersion Dispersion
1b 2b 3b 4) 5b
Unsaponifiable Unsaponifiable Unsaponifiable Unsaponifiable Unsaponifiable Unsaponifiable
Dispersion 6b Dispersion 7 b
Unsaponifiable Unsaponifiable
Dispersion 8 b
Unsaponifiable
Dispersion 9b
Unsaponifiable
2,400 1,720 1,680 2,215 1,665 1,540 190,100 190,300 189,300 191,000 181,000 21,000 20,580 20,680 20,460 18,700 245,500 245,300 216,400 217,000 269,000 7,590 8,570 8,510 8,900 6,700 6,530 6,410 7,230 7,330 7,330 7,590 9,850 10,030
1,670
...
..I
... ... 1,790
1,735 1,860
3,980 3,180
1.32 0.49 1.27 0.45
...
1,500 1,940 1,700 202,200 203,300 198,000 199,000
3,960 3,380
1.27 0.50 1.45 0.46
... ...
213,200 219,600 211,400 213,200 210,000 28,980 29,160 30,440 30,280 30,200
0.78 0.77 0.77 0.76 0.76 1.33 1.33 1.30 1.20 1.29
0.50 0.50 0.50 0.50 0.47 0.43 0.42 0.42 0.37 0.40
...
...
,.
..
... 19,480 20,270 19,400 18,510
... 250,000 250,000 232,000 224,900 ... 7,760 8,520 8,230 8,630 6,670 6,820 6,410 7,590 7,695 7,865 7,695 10,250 10,250
... 240,400 235,000 300,000 10,100 11,050 10,980 11,075 9,040 9,000 8,740 10,050 10,100 10,100 10,020 11,500 11,580
0.74 0.74 0.68 0.86 0.84 0.83 0.82 0.87 0.87 0.93 0.84 0.84 0.84 0.84 0.86 0.87
0.54 0.54 0.52 0.54 0.52 0.54 0.54 0.55 0.55 0.60 0.54 0.54 0.54 0.54 ,.
..
...
...
... ... 157,000
... .. .. ..
15,000
... ...
...
279,000
... ... ... ... ... ..
Table I1 shows the total amount in micrograms of vitamin A per ml. of standard and diluted fish liver oil taken for the determination of the recovery and the micrograms of vitamin A per ml. that were found. The per cent difference of these two values is shown in the last column. On the bottom of the table is sho\vn the mean per cent error of the recovery by the two methods on the whole and unsaponifiable fractions and the average error of a single determination for the two methods. The low values of the recovery errors point to the validity of the antimony trichloride and activated glycerol dichlorohydrin methods in the authors' hands. I n Table I11 are shown the results obtained on various fish liver oils in U.S.P. units of vitamin A per gram of oil by the activated glycerol dichlorohydrin, antimony trichloride, and ultraviolet absorption methods, Extinction ratios are also given. There are also presented the antimony trichloride, E:?&, X 2000, and multiple level bioassay values found by the Food Research Laboratories on the oils which they supplied, The antimony trichloride method employed by the Food Research Laboratories uses the increment procedure method (4), and the determinations yere not made a t the same time as the authors'. Considering these factors, the results are in good agreement. The one notable exception is on oil F where the authors' antimony trichloride and E:?&. values are about 20% lower than theirs. The fact that the authors' extinction ratio a t 300 mp was 0.74 compared to their value of 0.68 indicates that considerable oxidation of the oil had taken place between the time of their analysis and the authors', On the whole, the agreement between the values obtained by the activated glycerol dichlorohydrin and antimony trichloride methods is dose. However, almost all the values obtained by the ultraviolet absorption measurements are higher than the corresponding values obtained by the activated glycerol dichlorohy-
...
...
.. ... .... . ... I . .
Food Research Laboratories Long Island City, N. Y . The authors ar8( indebted to Daniel Melnick for these s a m d e s and determinations. b Aqueous dispersions of vitamins including vitamin A.
V O L U M E 19, NO. 2, F E B R U A R Y 1 9 4 7 drin and antimony trichloride reactions,
ill
The higher the extine-
300 tion ratio - mp of the oil, the greater the deviation of the ultra328
Table IV. Per Cent Deviations of SbC13 and E:& X 2000 Values on Whole Oil from Glycerol Dichlorohydrin
violet absorption value from the other two values. This relationship is implied in the studies of Oser et al. ( 5 ) . The whole oils B and C gave atypical colorimetric reactions -when assayed. Both oils gave violet instead of blue colors with -the antimony trichloride reagent, and the density of the violet color given with glycerol dichlorohydrin slowly increased from 2 t o 10 minutes instead of becoming constant a t the end of 2 minutes. This behavior is probably due t o the increased ratio of interfering substances for a given amount of vitamin A in the analytical sample. The unsaponifiable fractions of these oils gave t h e typical colorimetric reactions with antimony trichloride and activated glycerol dichlorohydrin. That the differences between t h e whole oils and the unsaponifiable fractions are not due to analytical errors was borne out by the similar results obtained on t h e same oils a t a later date. There are presented in Table IV the per cent deviations of the antimony trichloride and E::& X 2000 values on the whole oils from the glycerol dichlorohydrin values on the whole oils. The average per cent deviation of the antimony trichloride method was f14.58 and for the E;?& X 2000 64.53. If the deviations o n oils B and C (both of which gave atypical color reactions) are #omitted, the agreement between the antimony trichloride and ultraviolet absorption methods with the glycerol dichlorohydrin method is much closer, the average deviation of the antimony X trichloride method being -1.62% and that of the E:?;, 2000 being +17.11%,. In Table V are shown the per cent deviations of th(, antimony trichloride and the ultraviolet absorption values on the unsaponiBable fractions of fish liver oils from the glycerol dichlorohydrin values on the unsaponifiable fractions of the same oils. The agreement between the antimony trichloride and glycerol dichlorohydrin method is close, the average deviation of the antimony trichloride values being -4.11% and of the E;?&, X 2000 values +26.2570. TYhole oil estimations were made in order to determine to what extent those substances which interfere in the whole oil determinations by the antimony trichloride and ultra\lolet absorption methods interfere in the glycerol dichlorohydrin method. I n Table VI are presented the deviations of the whole oil values from the values obtained on the unsaponifiable fractions for each method. On the average, the values obtained by the glycerol dichlorohydrin method on the whole oils were 2.52yGlower than the unsaponifiable values, while for the antimony trichloride and ultraviolet absorption methods the whole oil values were about 7.5% higher. A conclusive statement regarding the significance of the glycerol dichlorohydrin method on whole oils cannot be made because of a n insufficient number of whole oil determinations, the emphasis i n this study being on the unsaponified fraction. On those whole oils which were analyzed by the glycerol dichlorohydrin method, the trend is for the whole oil values to be closer to the unsaponifiable values than in the other two methods. It is hoped that further investigation with a wide variety of oils will clear up this point.
(Whole oil value taken as 100)
+
SUMMARY
Activated glycerol dichlorohydrin has been used as a colorimetric reagent for the quantitative estimation of vitamin A in fish liver oils. Both the whole and the unsaponifiable fraction of the oils mere used, with emphasis on the unsaponifiable fraction. The new method was evaluated by comparing the results against the Carr-Price reaction and the ultraviolet absorption method. Taking the values obtained by the glycerol dichlorohydrin method on the whole oils as 100, it was found that the antimony trichlo-
Oil Sample
Deviation from G D H Value E:z&,(X328) SbCla X 2000
GDH Method U.S.P.
Oil B Oil C Oil B Oil C Mean deviation, % ' Average error, 70
44,850 1,350 124,350 60.700 9,995 125,900 202,750 19,875 252,260
E328
70
70
u./g.
1. Nonpercomorphum 2. Cod liver oil 3 . High shark 4. Hgalibut 5 . Hih D 6 . Argentine shark Oil D Oil E Oil F hlean deviation, Average error,
Extinction Ratio, E300
f 0.78 -22.44 9.37 f 0.08 -12.31 f14.30 6.19 4.60 - 2.83 * 8.10 1.62 Oct. 22, 1945 1,525 4-52.15 1,320 +65.90 April 1, 1946 1,735 +38.34 1,500 4-47.69 121.30 f14.58
+-
+ 349 .. 21 96 C 4.54 + 2.88 + 9.55 f 17.00 + 6.73 4- 52.75
-
7.11 +* 117.11
0.66 0.85 0.68 0.63 0.72 0.76 0.78 1.33
f
+
....
+158.40 +189.70
1.28 1.31
f129.40 +160.00 A 64.53 64.53
1.32 1.27
+
Table V. Per Cent Deviations of SbCl and E t Z . X 2000 Values on UnsaponifiableOil from Glycerol Dichlorohydrin (Values on unsaponifiable oil taken as 100) Extinction Ratio, E300 E328
Deviation from G D H Value E:E,(X328) SbClr X2000 Method Method
Oil SamDle
GDH Method U.S.P.
1. Nonpercomorphum 2. Cod 3. High shark ' 4. Halibut 5 . High D Oleum percomorphum Vitamin A in sesame oil Oil A
45,700 1,535 129,500 63,750 9,570 58,995 32,925 194,850
-
U./P.
Oil B Oil C Oil B Oil C Oil D Oil E Oil F Dispersion l a Dispersion 2 0 Dispersion 3' Dispersion 4a Dispersion 55 Dispersion 6" Dispersion 7a Dispersion 8" Dispersion 9'
-
5.14 6.19 4.56 6.20 4.18 2.90 2.37 -11.67
f 1.51
f 1.38
0.68 0.70 0.71 0.63 0.67 0.68 0.69 0.62
f62.11 +63.83
1.18 1.26
+- 40 .. 08 49
- 2.43 4- 6 . 3 8
+- 31 .. 61 01
Oct. 22, 1945 - 4.46 2,020 1,880 3.19
-
April 1, 1946 1,860 - 8.60 1,820 -13.75 198,500 - 4.21 18,955 8.52 228,450 - 5.14 7,760 2.19 8,520 f 0.59 8,230 i3 . 4 0 8,630 3.13 6,745 1.93 6,410 0.00 7,640 - 4.74 7,780 4.21 10,250 3.03
+70.98
1.27 1.45 0.77 1.25 0.74
-
1-29.70 4-33.42 +28.33 +33.73 +36.35 +31.82 4-29.30 f12.59
0.84 0.83 0.82 0.87 0.93 0.84 0.84 0.87
- 44 .. 71 61
b26.40 +26.25
++ 8 56..7905 +60.17 + 4.05 +23.17
+
-
+-
Mean deviation, % Average deviation, %
A
0.86
a Aqueous dispersion of vitamins including vitamin A.
Table VI. Per Cent Deviations of Whole Oil Values from the Unsaponifiable Values on Fish Liver Oils Oil Sample Sonpercomorphum Cod liver oil High shark Halibut High D
1. 2.
3. 4. 5.
Oil Oil Oil Oil Oil
B C D E F
Average error, %
SbClr Method 4.48 -29.12 + l O .1O. O 59
GDH Method
-
+
+
+
4.42
April 1, 1946 +41.20
+ 0.03 4- 1 . 6 9 +38.50
4-13.22
+ 7.72
-
1.86 -12.06 3.98 - 4.79 4.44
-
6.72
++- 1 724. 5.. 81924
+10.42
-
2.52
E1 % 1cm. X 2000 Method 1.06 +13.04 0.43 0.40 7.50
+ + ++
4-25.10 +17.16 01 . 09 0 3
+
...
+ 7.40
ANALYTICAL CHEMISTRY.
112 ride values were 1.637’i lower while the E;:; X 2000 were 17.11% higher (except in two cases which gave atypical colors). On the unsaponifiable fractions the per cent deviations of the antimony trichloride values were 4.11% lower and the E;?&, X 2000 values 26.25Y0 higher. Since there is good agreement between the values obtained by the glycerol dichlorohydrin and antimony trichloride methods, and since activated glycerol dichlorohydrin possesses many advantages over the Carr-Price reagent, the use of activated glycerol dichlorohydrin in the determination of vitamin il in fish liver oils is recommended. ACKNOWLEDGMENT
The authors are indebted to Daniel Melnick of the Food Research Laboratories, R. H. Kreider of Mead Johnson and Company, and S. M. Gordon of Endo Products, Incorporated, who supplied the samples used i n this study.
LITERATURE CITED
(1) Gridgeman, N. T., “Estimation of Vitamin A”, pp. 11-19. Lever Bros. and Unilever Limited, Port Sunlight, Cheshire England, 1944. (2) Oser, B. L., Oil,Paint Drug R e p t r . , 139,4 (1941). (3) Oser, B. L., Melnick, D., and Pader, M., IKD.ENG. CHEM.. ANAL.ED.,15,717 (1943). (4) Ibid., 15, 724 (1943). (5) Oser, B. L., Melnick, D., Pader, M.,Roth, R., and Oser, M., Ibid., 17, 559 (1945). (6) Rawlings, H. W., and Wait, G. H., Oil and Soup, 33,83 (1946). (7) Sobel, A. E., and Werbin, H., IND.ENG.CHEM.,ASAL.ED.,18, 570 (1946). (8) Sobel, A. E., and Werbin, H., J . Biol. Chela., 159, 681 (1945). (9) Twyman, F., and Allsopp, C. B., “Practice of Absorption Spectrophotometry”, 2nd ed., p. 57, London, Adam Hilger, 1934. (10) Vandenbelt, J. M., Forsyth, J., and Garrett, A., INP. ENQ. CHEM.,ANAL.ED. 17,235 (1945). (11) Wilkie, J. B:, J . Assoc. Oficial A g r . Chem., 28, 547 (1945). PRESENTED before the American Society of Biological Chemists, Atlantio City, X. J. This study was aided by a fund granted by the American Home Products Corporation, and by a grant from the Cnited Hospital Fund of the City of Greater New York.
Analysis of Mixtures of Mercurous and Mercuric Mercury and Sulfuric Acid BENJ. WARSHOWSKY AND PHILIP J; ELVING, Publicker Industries, Znc., Philadelphia, Pa.
A procedure is described for the determination of mercurous and mercuric mercury and of sulfuric acid in mixtures such as are found in used hydration catalysts of the sulfuric acid-mercuric sulfate type. Afercurous mercury is determined iodometrically, total mercury after cerate oxidation by thiocyanate titration, and sulfuric acid by neutralization in the presence of large amounts of chloride.
A
SOLUTION of mercuric sulfate in sulfuric acid is often used as a catalyst’ for the hydration of organic compounds, as in the addition of water to unsaturated carbon to carbon linkages. After some use, part of the mercuric salt is reduced to mercurous sulfate which is catalytically inactive. It is desirable, therefore, to be able to determine rapidly and simply the concentrations of mercurous and mercuric mercury in the catalyst solution, so as to know when the catalyst needs regeneration and the extent of regeneration necessary. Since the sulfuric acid concentration must be carefully adjusted for the particular hydration reaction used to obtain optimum yields, a rapid method for its determination in the presence of the mercury salts is necessary in order to keep the concentration of acid at the proper level. The solution of the problem was found by adapting known methods for the determinat,ion of mercurous and mercuric mercury, and devising a simple procedure for the determination of the sulfuric acid, I n a solution containing mercurous and mercuric mercury and sulfuric acid, the total mercury content’ is first determined by oxidizing the mercurous mercury to mercuric mercury with a sulfato-ceric acid solution and titrating the resulting mercuric ions with standard thiocyanate solution (2, 4 ) . The mercurous mercury is determined in another sample by oxidizing it to the mercuric iodide complex with iodine liberated from an excess of standard iodate solution and determining the excess iodine by titration with standard thiosulfate solution (1, 3 ) . Sulfuric acid is determined by titration with standard sodium hydroxide solution in the presence of excess sodium chloride. DISCUSSION
Willard and Young ( 5 ) describe a method for the determination of mercurous mercury in.the presence of mercuric mercury based
on the oxidation of the former in hot sulfuric acid solution by excess standard sulfato-ceric acid solution and titration of the excess electrometrically with standard ferrous sulfate solution. The authors of the present paper obtained poor and erratic results when the method was applied to synthetic mixtures. Since the values obtained depend on the reduction of ceric to cerous ions, any substance which is oxidized under the conditions used interferes with the determination; apparently, in the experiments made, some substance may have been present which erratically reduced ceric ions; Willard and Young indicated that large amounts of mercuric ion caused a slight decomposition of ceric ion in hot solution. Blank corrections were not determined. The possible presence of organic matter in actual catalyst samples was another reason for avoiding the use of a method for measurement which involved boiling with an acid ceric solution for 30 minutes, HoTvever, the procedure employing oxidation with sulfato-ceric acid was used as a preliminary oxidation step in the determination of the total mercury content, sirace in this case the results Tvere not based on the consumption of the oxidant, Pure samples of mercurous sulfate were analyzed by the iodometric method (3) and a precision of 5 parts and an accuracy of 2 to 3 parts per thousand were obtained. The presence of mercuric sulfate did not affect the results. Since the consumpt’ionof iodine is employed as a measure of the mercurous mercury content, any substance which reacts with the iodine or the thiosulfate under the conditions used will give erroneous results. Accordingly, i t is necessary t o check the procedure in the presence of any organic sludge that may be formed in the hydration reaction and be present in the catalyst sample. I n many cases extraction with organic solvents or simple distillation can be used to remove interfering organic materials.
