NOVEMBER 15, 1940
639
ANALYTICAL EDITION
volume of solution containing the quantities of reagents used in the test.
Sodium Oxalate ( I ) Test SULFATE.Ignite 10 grams in a platinum crucible, using an alcohol flame to avoid contamination from a gas flame. Take up the residue in 25 cc. of water and 3 cc. of bromine water. Heat for 15 minutes on the steam bath, neutralize with hydrochloric acid, and add an excess of 1 cc. of dilute acid (1 4). Heat to remove bromine, make to a volume of about 100 cc., heat to boiling, add 5 cc. of 10 per cent barium chloride solution, heat on the steam bath, and allow to stand overnight. If a precipitate is formed the weight of the ignited precipitate should not be more than 0,0005 gram greater than the weight of the ignited precipitate from a blank made Tith the quantities of reagents used in the test.
+
Sodium Thiosulfate (2) Requirement MATTER. Not more than 0.005 per cent. Test INSOLUBLE MATTER. KOchange except that the n-eight of the residue should not exceed 0.0005 gram.
ISSOLUBLE
Toluene (10) Requirement WATER. To pass test (limit about, 0.02 per cent). Test WATER. Place 10 cc. in a test tube (150 X 16 mni.) loosely stoppered. Pack in cracked ice. No turbidity should be observed a t the end of 3 minutes.
Xylene (10) Requiremerit WATER. To pass test (limit about 0.02 per cent). Test WATER. Place 10 cc. in a test tube (150 X 16 mm.) loosely stoppered. Pack in cracked ice. S o turbidity should he observed at the end of 3 minutes.
Zinc Sulfate ( 7 ) Test Dissolve 2 grams in 9 cc. of water and 1 cc. oi 80SITR~TE. d i m chloride solution containing 5 mg. of SaCl per cc. ;Idd 0.20 cc. of indigo carmine solution (1 in 1000) and 10 cc. of sulfuric acid. The blue color should not be completely discharged in 5 minutes.
Specifications Previously Published (1) Committee on Analytical Reagenta, IKD.ESG. CHEY.,17, 7.56 (1925).
( 2 ) Ibid., 18, 636, 759 ( 1 9 2 6 ) . (3) Ibid., 19, 645 (1927). (1) Ibid., 19, 1369 ( 1 9 2 7 ) . (5) Ibid., 20, 979 ( 1 9 2 8 ) . (6) Ibid., Anal. Ed., 1, 1 7 1 ( 1 9 2 9 ) . (7) Ibid., 2, 351 (8) Ibid., 3, 221 (9) Ibid., 4, 154 (10) Ibid., 4, 3 4 7 (11) Ibid., 5, 289
(1930). (1931). (1932). (1932). (1933).
PRESENTED in connection with the report of the C o m m t t e e on Analytical Reagents a t t h e 97th Meeting of the American Chemiod Society, Baltimore, Md.
Spectrophotometric Determination of Vitamin A Critical Study of Applicability to Fish Liver Oils D. T. EWING
AND
J. RI. VANDENBELT, Rfichigan State College, East Lansing, >rich.,
4. D. EMRZETT
AYD
ihn 0. D. BIRD, Parke, Davis & Company, Detroit, JIich.
I
T IS the general opinion that the determination of vitamin A in fish liver oils and other products by physical methods has shown definite promise of giving satisfactory results. The several factors in the spectrophotometric method have therefore been studied in an effort to increase our knowledge of its accuracy, in the hope that it may come into more regular application as a quantitative procedure. The amount of vitamin A in a fish liver oil may be estimated quantitatively in a number of m-ays. The most common are the biological, the colorimetric, and the spectrophotometric methods. The historical development of the subject has been covered very thoroughly by Munsell (6). Suffice i t to state that the first biological assays were carried out b y Drummond and Coward ( 2 ) ,the first color reaction using arsenic trichloride b y Rosenheim and Drummond (7), and the color reaction using antimony trichloride by Carr and Price (1). Spectrophotometric measurement in the visible range at 698 m p was made first by Drummond and Morton ( 3 ) . Takahashi et al. (8) first reported on the selective absorption characteristics of vitamin L4in the ultraviolet, while the absorptive maximum in that region was established a t 328 mp by Morton and Heilbron ( 5 ) . Comparison of the data by these methods has shown, in the hands of various workers, both small and large discrepancies.
Because it is generally recognized that the spectrophotometric method is intrinsically capable of making very accurate determinations with both organic and inorganic substances, the authors have made a careful study of the factors involved with respect to vitamin A in fish liver oils. This has included the solvent and its action, the effect of variation in cells, the application of Lambert’s and Beer’s laws, the importance of instrument adjustment, the light source, and incidentally the practical limits of a few interfering substances.
Method and Plan Having investigated the above factors, the next step was to test the precision of the technique. X series of many determinations was made on each of several fish liver oils to ascertain the spread or variation in the maximum extinction coefficient or E: yalue a t 328 rnF (325 to 328 m p ) on each sample. This was taken as an indication or index of the reproducibility of the technique. The spectrophotometric setup consisted oi a Bausch & Lonib sector photometer with a quartz optical system and a Bausch & Lomb medium quartz spectrograph. A condensed spark between tungsten steel electrodes supplied the ultraviolet radiation, the energy being generated by a Bausch B: Lomb 450 VA inductance transformer. Eastman S o . 33 photographic plates Fere
Fm,
INDUSTRIAL AND ENGINEERING CHEMISTRY
640
TLBLE I. EFFECTO F TnrE Time Elapsed after Solution
1 7 cni
.lf%71 3
15 30 60
Hours 29 7 28.1 26.6 25.6 25.7 23.2
2 3
$5 I .25 25 47
TABLE 11. COMPARISON OF EXTISCTION COEFFICIEKTS 13 I S O P R O P Y L .4iiD ABSOLUTE ETHYL ALCOHOL
Halibut (3898) Halibut (2458) Halibut (2668) Halibut (1528) Halibut (2978) Mixture (4255) Cod (9758)
31.5 26.8 31 . 0 31 3 31.8 78.7 1.59
31.6 27.2 32 0 30.9 31.6 81 4 1.6’2
1,003 1.015 1.032 0.987 0 994 1.034 1.019
used throughout. They were processed by S o . D-1 developer a t 18” C. Samples of the fish liver oils were weighed out and dissolved in redistilled isopropyl alcohol (Eastman). The spectrophotometric value was then determined. The first photographic exposure was made 10 minutes after preparation of the solution, and other exposures followed immediately. The plate was processed under carefully controlled and reproducible conditions. When the emulsion was completely dry, the “isodensity” or “reversal” points mere marked on the glass side. The log I o / I of the absorptive maximum was then determined, where Io equals incident light (100 per cent), and I equals per cent of light transmitted a t the wave length of the absorptive maximum. The extinction coefficient of the absorptive maximum was calculated from the formula: 1 ‘
1% cm. =
d X
~
1
x
VOL. 12, NO. 11
are given in Table 111. It is evident that the cells should always be checked against each other, in order to eliminate variations due to errors in the length of cells and differences in the absorption of the end pieces. Extreme cleanlinebs of the cell \Tall is essential, particularly TI ith reference to oily residues remaining from the previous .ample. Just before each run, the quartz surfaces should be TI iped n ith fresh lens paper. Perpendicularity of the ends of the cells to the light beam is necessary, and the cells must always occupy exactly the same position with respect to the optical path. LAJIBERT’SLam. A halibut liver oil sample (908,871) wa5 dissolved in isopropyl alcohol and diluted so that the a b s o r p tion of the solution was suitable for an determination in the 10-mm. cells. (The log Io/I was equal to approximately 1.0.) The E:?m. value of this concentration was obtained also in each of the cell lengths 1, 2.5, 5, 20, and 50 mm. The solvent path shutter readings were changed to conform n i t h the various optical densities of the different length cells. The E:?m, values (Table IV) determined under these conditions did not change with cell length, except for small variations ascribable to the method, thereby complying with the requirements of Lambert’s law. BEER’SLAW. B y proportionally increasing the concentration of the fish liver oil solution in the cells shorter than 10 mm., and decreasing i t for examination in the longer cells, the values (Table I T ) a t a wide range of concentration were obtained. Over a range of 50 times difference in concentration, the variation in values for the oil was no greater than that obtained when a series of a similar number of determinations was made a t one concentration and one cell length. Therefore, both Lambert’s and Beer’s laws are valid for dilutions of vitamin d within the accuracy of the method used. LIGHTSOURCE. TWOsources of ultraviolet radiation were used. The first was a condensed spark between wedge-tipped tungsten alloy steel electrodes. Using a slit width of 40 microns, the amount of light emitted by this spark gave, in 15
log ZolZ
70concentration
where d equals length of light path through the solution in cm., and the per cent concentration equals 100 times the weight of sample in grams divided by the milliliters of solvent.
TABLE111. COMPARISOS OF CELLS BY MEASUREMENT OF MOLECULAR EXTIKCTIOX COEFFICIEXTS OF POTASSIUM KITRATE c,
Solvent Cell B8:L.l B . & L. 2 Zeiss 1 Zeiss 2 Zeiss 3 Zeiss 4
Fundamental Factors EFFECTOF TIMEON E:?m, VALUE IK SOLUTIOK. Table I gives the values of halibut liver oil (3898) in isopropyl alcohol a t varying time intervals after addition of the solvent to the sample. The absorption reached a maximum value after 5 minutes and remained fairly constant for about a n hour, after which there was a significant decrease. COMPARISON OF SOLYEKTS. Table I1 gives extinction coefficients at 328 mw in isopropyl and absolute ethyl alcohol. The ratios, being near unity, show that these alcohols can be used interchangeably in the determination of the value of fish liver oils. Isopropyl alcohol was used as a solvent throughout this work because i t is a superior solvent for oils. This is a n advantage in assaying oils of low potency. INFLUEKCE OF TYPES, CLEANLINESS, AND POSITION OF CELLS. Bausch & Lomb 10-mm. cells were used in part of this work and Zeiss 10-mm. cells in the other part. I n order to ascertain the relative absorption of the end pieces of the several cells, the molecular extinction coefficient of potassium nitrate was determined in both types. The values obtained
Solution Cell BtLL.2 B.& L. 1 Zeiss 2 Zeiss 1 Zeiss 4 Zeiss 3
X = 301 mp
7.00 7.00 6.97 6.97 6.67 7.00
TABLE IT’. -4PPLICATIOX O F LAMBERT’S AND BEER’SLAWE. (Halibut liver oil No. 908,871) Length of Cell 1 2.5 5 10 mm. mm. mm. mm. 7
Concentration, c. IC
0.314 0.12i
0.063 0.0314
30.1 29.9 29.1 30.1 28.3 28.8
..
0,0157 0,0063
E:?~, Values
7
..
Summary (40 tests) :
28.1 28.1 28.7 28.2 29.2 29.6 28.1 29.5
28.9 29.1
..
..
27.7 2s 2
..
.. ..
..
50 mm.
.. ..
..
-.--.
..
.. 28 9 2s 9 29.8 29.8
20 mm.
..
.. 29.6 29.6 29.7 29.1 29.6 29.9 29.1 29.1
Mean l l a x l m u m range, 70 Maximum deviation,
70
..
..
28.5 29.8 29.7 29.1 28.0 28.4
28.3 27.7 29.9 29.9
29 04 S 3 4 8
..
ANALE-TICAL EDITION
NOVEMBER 15, 1940
TABLEv.
Test S O .
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 16 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
H A L I B ~LIVER T OIL 3898 328 mp
VALVES O F
(As determined by t h e spectrophotometer) ConcenSpectroLog Io/I ShutFer tration, gram 328 mp Reading NO. 70 390a 390b 391a 391b 392a 392b 393a 393b 394a 394b 395a 395b 396s 39613 397s. 397b 398a 398b 399s 399b 400a 400b 401a 401b 402a 402b 403a 403b 404a 404b 405a 405b 406a 406b 407a 407b 408a 40Sb 409a 409b 410
0.0330 0.0336 0.0300 0.0304 0.0314 0.0320 0.0306 0,0292 0.0318
9-10 9-10 8-9 9-10 11-12 10-11 11-12 11-12 10-11 9-10 10 9-10 10-11 10-11 12 11-12 10-11 10 9-10 9 11-12 11-12 9 9-10 11-12 12-13 11 10-11 11-12 11-12 12 11-12 12-13 12-13 10-11 10-11 11-12 11-12 12-13 12-13 10-11
.4T
E1 l% cm.
1.02 1.02 1.07 1.02 0.94 0.98 0.94 0.94 0.98 1.02 1.00 1.02 0.98 0.98 0.92 0.94 0.98 1.01 1.02 1.05 0.94 0.94 1.05 1.02 0.94 0.90 0.96 0.98 0.94 0.94 0.92 0.94 0.90 0.90 0.98 0.98 0.94 0.94 0.90 0.90 0.98
30.9 30.9 31.8 30 1 . 43 32.6 30.9 30.9 31.2 32.5 31.2 31.9 32.0 31 2 . 50 32.2 30.8 31.8 30.5
641
stearic acid, has an E!?m, value of but 0.03, which is of no practical consequence. The metallic salts of fatty acids, for the most part, are insoluble in isopropyl alcohol, and are eliminated thereby. Copper oleate, although slightly soluble, has a deep green color, and can be easily detected. On the other hand, ferric oleate is soluble in isopropyl alcohol and has significant absorption a t 328 mp a t concentrations as lorn as 0.001 per cent. It can be detected by means of the thiocyanabe test when present to the extent of only 0.0005 per cent. It is obvious value that if the chemical test is negative for iron, the E:?mm. has not been significantly increased.
Application of Spectrophotometer to Fish Liver Oil
SPECTROPHOTOMETER SERIES. Table V gives all values obtained with the Bausch & Lomb spectrophotometer in a rep3 1 . 54 0.0298 31.5 resentative series of tests on halibut liver oil 3898, this being 32.6 0,0322 typical of the seven fish liver oils studied in the long series 31.6 0.0290 on reproducibility of technique. The data show, respectively, 3 21 . 40 30.8 0.0312 the test number, spectrogram number, solution concentra31.4 tion, shutter reading of the “isodensity” point a t the 328 mp 31.3 0.0300 absorptive maximum on the finished spectrogram, log lo/l 3 1 ..73 0.0290 32.4 value or solution density corresponding to the shutter reading 31.2 0 0288 a t the wave-length maximum, and the calculated exbinction 31.2 0.0304 32 2 .. 22 coefficient a t 328 mp. 3 0.0302 The summary at the bottom of Table V shows that the 31.1 31.1 30.5 0.0296 maximum range for these 41 tests was 7.0 per cent-that is, 30.5 the difference between the highest and the lowest values was 0.0308 31.8 7.0 per cent of the mean. The maximum deviation, or largest 91 A R Summary (41 tests) : Mean value Maximum range, % 7.0 per -cent difference between any single value and the mean, 3.5 Maximum deviation, 5 was 3.5 Der cent. Table-VI gives the spectrophotometric E:?m. values obtained for the other oils studied in the long series. On account of the space required, it is not possible t o present the detailed seconds, a suitable exposure at 328 n l p for an Eastman 33 plate, data, as in the representative series of halibut liver oil 3898. if the optical density of the absorbing solution was equal to approximately 1.o. ~ ~ a ~ ~ i hydrogen ~ l ~~ discharge ~d ~, It is easy t o note, hoTJ-ever, the excellent reproducibility and the total absence of eccentric values, although the oils extube T\-as Although requiring somelThat longer exa mide range Of potency. amined posure time than the spark, it was found to be superior from ISFLUENCE O F XUMBER OF DETERMINATIOKS ON REPREthe standpoint of both convenience and reproducibility, In SENTATIVE 3 I E A N VALUES. It is obvious that all the either case only jvhen the various parts of the spectrophotoE:&, values for halibut liver oil 3898, presented in the last metric setup were in exact optical alignment vias it possible to obtain optimum working conditions as to minimum exposure time and transmission into the lorn ultraviolet region. TABLEVI. VALUESOF FISHLIVEROILS AT 328 mp EXPOSUREL.4TITUD.E AXD DEKSITYOF ( a s determined by t h e spectrophotometer) PHOTOGRAPHIC PLATE.The- processing of Cod Halibut Mixed Halibut Mixed Mixed Cod---“Konsap.” the photographic plate was carried out under (16,519) (4288) (16,319) (18,869) (16,949) (9758) (9758) well-defined conditions. Using an Eastman 77.6 78.3 125 129 1.57 1.34 1.56 1.56 50.7 49 0 15.0 39.7 calibrated 21-step tablet an exposure by 74.5 7 i . 6 40.5 50.5 49.0 14.8 1.56 1 . 5 3 130 129 1.57 1.31 7 5 . 2 7 9 . 4 138 3 9 . 8 51 0 4 8 . 7 1 5 . 2 130 1 . 6 7 1 .32 direct contact was made on each plate. The 78.4 7 8 . 4 1 . 5 71 1 . 5663 1 . 6 4 40.5 51 .o 1.31 50.7 15.4 127 134 129 4 0 . 2 4 9 . 6 127 7 9 . 8 7 8 . 4 1 . 5 4 1 . 6 0 5 1 . 0 1 . 5 5 1 .29 1 5 . 2 density us. log exposure curve gave a gamma 82.0 7 9 . 0 1.51 1.60 39.2 49.5 1.55 1.31 48.7 14.8 127 125 of 0.9 for the plate as processed under care1.67 1.28 81.0 79 0 49.0 127 125 1.59 1.60 40.8 50.7 14.8 50.7 74.8 79.7 40.0 51.0 129 132 1.59 1.56 1.65 1.37 15.0 fully controlled conditions. The isodensity 1.65 1.37 51.0 49.6 7 9 . 4 79 4 1.56 1.60 40.0 15.0 129 126 51 . o 7 8 . 0 79.9 1.67 1.35 40.8 1.63 1.60 50.0 15.3 129 132 points had a value of approximately 0.6 51.0 79.6 75.4 126 132 1.63 1.37 39.2 1.63 1.61 50.0 l5,4 as read on an Eastman transmission den51.0 79.6 79.0 1.63 1.35 1.59 1 . 5 8 39.2 51.0 14.8 129 128 126 126 1.63 1.37 49.0 78.9 79.8 1.53 1.57 40.0 50.5 15.0 sitometer. 1.66 1.41 78.9 78.3 1 . 5 6 1.57 49.0 39.2 49.0 14.8 129 128 1.59 1.41 51.2 80 8 8 0 . 0 1.55 1.63 40.0 49.8 15.4 128 131 INTERFERING SVBSTASCES. Some few 1.60 1.41 77.6 81.7 1.63 1.62 51.2 50.5 15.3 127 126 39 2 compounds commonly found with fish liver 1.55 1.41 78.3 80.0 1.56 1.57 50.2 39.2 49.0 15.4 131 127 1.57 1.40 7 5 . 3 79 0 .. , 1.56 1.53 51.2 49.8 127 134 ... oils exhibit absorption a t 328 mp, Naturally 1.63 1.41 77.6 7 9 . 0 ... ... 1.63 1.55 49.0 49.8 127 129 1 . 5 7 1.26 1 . 6 3 1 64 127 50.0 8 0 . 9 78 3 . . . . . . 131 50.8 occurring are the unsaturated long-chain 1.59 1.28 49.2 ... ... 1.52 1.57 131 133 75.3 76.7 . . fatty acids, oleic and palmitic, which have 1.63 1.28 1.55 1.64 ... (8.2 79.0 . . . ... ... ... 1.59 ... ... ... ... 1 . 5 8 1.62 ... 81.6 80.7 . . . extinction coefficients of 0.88 and ... 1 . 5 1 1.58 1 . 6 2 ,.. .. .. ... ,.. ... ... .. .. ,. ... ... 1 . 5 6 1 57 1.59 ... .. . 0.63, respectively. I n low potency oils, such ... ... .. .. ... ... 1.60 1.57 as cod liver oils, these should be removed by Mean 50.12 78.68 128 6 15 05 39 85 1 589 1 346 saponification. The saturated compound, (6)
0 0334
Y I . _ j .
r--
INDUSTRIAL -4ND EXGrINEERISG CHEMISTRY
642
TABLE
‘rest
KO.
5 6 8 9 10 11 12 13 14 15 16 17
18
19 20 21 22
VII.
through the solution. E’iom the scale leading, calibrated a- the l o g l o / l , the value of the ianiple can he calculated.
VALUESO F HALIBUT LIVERO I L 3898 AT 328 mp
(Determined by the vitameter) --Operator 1--Operator Concentra- Vitameter Vitameter tion, male scale % reading 1 cm. reading 0.0222 0.713 32.1 0.756 0.0211 0.690 32.7 0,701 0.0189 0.663 35.1 0.672 0.0211 0.701 33.2 0.713 0.634 31.7 0,661 0.0200 0.0222 0.697 31.4 0.677 0.0211 0.670 31.8 0,639 0.0189 0.591 31.3 0.640 0.0189 0.648 34.4 0.659 0.0222 0.699 31.4 0.692 0.590 31.2 0.642 0.0189 0.0211 0.693 32.8 0.670 0.0200 0.639 31.9 0.632 0.0189 0.591 31.3 0.643 0.0222 0.681 30.7 0.677 0.0200 0.600 30.0 0.641 0.0222 0.689 0.690 31.0 0.0211 0.669 31.2 0.614 0.0200 0.641 32.0 0.648 0.0189 0.600 31.7 0,610 0.0200 0.670 0.634 33.5 0.670 31.7 0.686 0.0211
Summary (22): Mean Maximum range, $& hhximum deviation,
76
32.00 15.0 8.8
Summary (44): Mean 32,20 Maximum range, yo 19.8 .\laximum deviation, yo 1 0 . 3
J’ITAMETER SERIES. U s i n g thi3 instrument, several zerieb of determinations mere made on fish liver oil samples corresponding to those run on the spectrophotometer (Table Y)for oil 3898. Table VI1 contains the results obtained with the vitameter on this same oil. These include the test number; the per cent concentration; the scale reading, representing the average of 10 observations on each dilution, made value for by two operators; and the calculated E:m, each series of 22 tests. (The authors gratefully acknowledge the assistance of Cyril1 J. Campbell for his cooperation in the vitameter determinations.) The means of the values by the two opeiators, working independently and on unknown concentrations, are not significantly different. The mean values by operators 1 and 2 are 32.00 and 32.40, respectively, with a difference of only 0.40 or 1.2 per cent of the mean. IXFLUEKCE OF KUMBER OF DETERMINATIONS ON ACCURACY OF J-ITAMETER MEANS. Table VI11 shows the accuracy of vitameter results obtained for six fish oils when the values fiom differing numbers of independent runs were averaged to give an value for an oil. The last column gives the value obtained by a single determination on each oil. In parentheses, the percentage differences between these single T-alues and the means of 18 determinations are given. The other columns contain similar data for the means of larger numbers of determinations. The mean of a number of runs becomes, in general, more nearly that of the mean of 18 as the number increases. This is best illustrated by the means of the per cent differences (at the bottom of the table). These values decrease from 5.37, in the case of the single determinations, to 0.69 in the case of nine determinations. The maximum per cent differences decrease in the same fashion-from 11.50 to 1.11. The maximum per cent difference for six determinations is 1.79. This value, being within experimental error, establishes this number (6) as the minimum number of tests for routine laboratory practice. METHOD ADOPTED.I n the light of observation the following procedure has been used in routine analysis.
2-
El% 1 cm. 34.1 33.2 35.5 33.8 33.1 30.5 30.3 33.9 34.8 31.2 34.0 31.7 31.6 34.0 30.5 32.0 31.1 29.1 32.4 32.2 31.7 32.4 32.40 19.8 10.2
...
... ...
column of Table V, are in very good agreement. This becomes even more evident if the series is divided into smaller groups. By comparison of the means of these successively smaller groups with the mean of the whole series, i t is possible to ascertain the number of determinations necessary for a value of the oil. satisfactory representative mean For example, if the first 20 values (chronologically obtained) are averaged, the mean is 31.44, which is only 0.06 per cent lower than the mean of all the values. The mean of the second half of the series, 21 values, is 31.49, but 0.10 per cent above the mean of all. The means of successively smaller groups of E: values show slight though increasingly larger deviations from the total mean. If six values be taken in each group, the maximum difference of any mean from that of the whole series is 1.46 per cent. If the mean of the two values from a single plate be taken, the largest difference from the total mean is 3.05 per cent; the mean of these per cent differences being 1.33. This demonstrates that a very satisfactory representative value for an oil can be obtained from the mean of but two spectrophotometric determinations.
Tm,
Application of Hilger Vitameter to Fish Liver Oils In addition to the study of vitamin A by means of the ultraviolet spectrophotometer, a similar investigation was made with the Hilger vitameter, an instrument developed for the express purpose of determining vitamin A in fish liver oils. A ray of ultraviolet light from a copper arc first passes through a filter transmitting pri%cipally copper %ave lengths 3247.55 A. and 3273.97 A., and then through a solution of fish liver oil in isopropyl alcohol. The ray impinges on a fluorescent screen as a line of light approximately 1 cm. in length. The intensity of this line is compared visually with the intensity of a similar line from a corresponding ray which does not pass
TABLE VIII.
VOL. 12, NO. 11
Duplicate samples (0.20 to 0.25 gram) of a fish oil are neighed on the analytical balance into SO-ml. flasks. To each is added from a buret a volume in milliliters of isopropyl alcohol equal t o 100 times the weight of sample in grams. This makes a
INFLVENCE O F S U M B E R OF DETERMINATIONS O X a~CCURACY OF
VITAMETER MEANS
7
18
Type of Fish Liver Oil
Halibut (3898) Mixture (25,679)
23.50
Halibut (25,379)
30.71
Distillate (25,709)
...
... ... 33.14 ... 120.4 ...
Sumber of Determinations 6 3 2
1
>lean E ; ? ~ , Values ~-
7 -
31.96
Halibut (25,319)
9
32.63 (4-0.67) 23.34 (-0.68) 31.05 (tl.11) 33.15 (4-0.03) 121.4 (+0.83) 147.6 (+0.82)
32.70 (tO.74) 23.38 (-0,51) 31.10 (f1.27) 32.58 (-1.79) 119.2 (-1.00) 147.7
33.30 (f1.34) 23.93 (+1.83) 29.23 (-4.82) 33.07 (-0.21) 127.0 (+5.31) 147.3 (f0.62)
7
32.40 (f0.44) 25.40 (f8.10) 29.25 (-4.76)
32.1 (tO.14; 26.2 (f11.50) 28.8 (-6.23)
32.10 (-3.14)
32.3 (-2.54)
123.5
131.0 (+8.81) 142.0 (-3.01)
(+2.58)
Tuna concentrate (25,819)
146.4
Average of per cent differences Maximum per cent difference
...
0.69
1.03
2.35
3.50
5.37
...
1.11
1.79
5.31
8.10
11.50
...
(+O,SB)
113.5 (-1.98)
ANAL1 TIC \I2 EDITIUA
NOVEMBER 15, 1940
weight-volume concentration of 1.00 per cent. Further preliminary dilutions are made with calibrated pipets and small volumetric flasks until the concentration necessary to give a vitameter scale reading extinction between 0.50 and 0.75 has been established. Three suitable dilutions are then prepared for each sample. The mean of ten independent scale readings is taken as the vitameter extinction value for each dilution. Six E:?m. values are calculated by dividing the vitameter extinction values by the per cent concentrations of the corresponding solutions. The mean of all six Et?m. values thus obtained gives a satisfactory determination of the vitamin A content of the oil. The nonsaponifiable matter was prepared according to the procedure given by Morton (4).
Comparison of Methods COMPARISOK OF SPECTROPHOTOMETER AND VITAMETER E:?m. VALUES. Thus far the work has dealt extensively with fundamental factors in the determination of vitamin A by means of the spectrophotometer and the vitameter. The degree of precision and reproducibility of both methods has been demonstrated in the series of 41 and 44 values, respectively, with spectrophotometer and vitameter, on halibut liver oil 3898 (Tables V and VII). Because of the space required, i t is impossible to present the detailed data for each oil studied in the several series. I n Table IX, however, the summary data for both methods are presented. This includes the number of tests, the maximum range, the maximum deviation, the mean E:?",. value for each sample, and in the last column the ratio of the vitameter to spectrophotometer mean. It is evident, from the maximum range and maximum deviation data, that the vitameter is much less precise in its operation than is the spectrophotometer. Including the values for the cod liver oil and its unsaponifiable fraction, the average maximum range for the vitameter is about tn-o and one half times that of the spectrophotometer. Despite these wider ranges, the mean values by the tTVo instruments on these long series are in good agreement, showing that a sufficient number of values have been obtained to approximate a normal distribution. The two instruments must be measuring, therefore, the same significant characteristic of the oils.
TABLE X. COMPARATIVE
VALFESOF FISHLIVEROILS AT 328 mp
(Determined b y the spectrophotometer and vitameter)
Fish Oil S o . 30,839 10,358 10,358 "nonsap." 13,448 36,470 "nonsap." 13,248 32,539 32,549 32,469 32,509 32,419 32.489 32,499 32,519 32.479 32,369 32,459 32,379 32.529 32,389 32,139 32,409 32,399 32,589 32,449 32.429 32,559 32,579 32,599 2,668 3.258 3,658 2,978 32,569 15,768 17.789 17,779 13,108 11,598 31,599 13,438 14,748 17,849 8,008 8..548 8.5.58
8,048
COMPARISONOF TABLE Ix.
SGllll1RY
VhLUES O F F I S H
LIVER OILS
AT
325 rng (Determined by t h e spectrophotometer and vitameter) Maxi1Iaximum E1 R Sumher mum Devia1 em. of Range, tion, Mean Ratio, Tests % 70 values V/Sa Type of Fish Liver Oil Halibut (3898) W 5 41 7.0 3.5 31.46 1.023 19.8 10.3 32.20 (V) 44 Halibut (16,519) ( S ) 41 5.0 2.8 50.12 1.005 12.0 50.39 ( V ) 40 22.2 Mixed (4288) ( S ) 46 8.9 5.1 75.08 1.014 ( V ) 46 17.8 9.0 70.82 ( S ) 42 Llixed (16,319) 7.0 4.2 128.6 1,031 21.0 11.0 132.6 (VI 40 Halibut (18,569) (SI 17 4.0 2.3 15 05 1.005 18.5 (VI 30 10.6 15.12 ( S I 17 Mixed (16,949) 4.0 2.0 39.85 1.005 21.2 13.8 40.07 ( V ) 30 Cod (9758) ( S ) 77 10.0 5.0 1.589 0,909 (VI 110 18.0 10.4 1.444 ( S ) 22 Cod "nonsap." (9758) 11.2 6.7 1.346 1,017 ( V ) 62 16.9 8.8 1.369 Av. (8) (SI 7.1 3.9 ( 17) 1g.4 10.7 a
S, Spectrophotometric
values.
V , Vitameter Et?m, values.
643
E:& a t 328 mp Spectrophotometer Vitameter Cod Liver Oils 0,909 1.29 1.25 1.41 1.40 1.44 Halibut Liver Oils 13.4 14.6 16.6 16.7 16.8 17.3 18.8 18.8 19.0 19.3 19.5 20.3 20.6 21.4 21.9 22.4 23.1 25.5 25.8 25.9 28.8 29.2 29.3 31.0 31.3 31.6 31.8 32.5 36.8
0,884 1.34 1.28 1.41 1.46 1.40
Ratio,
v/s
0.973 1.039 1.024 1.000 1.043 0.972
13.7 15.2 16.2 17.1 17.2 17.3 18.5 18.8 19.6 18.9 19.4 20.9 20.0 21.4 22.0 22.7 23.9 25.2 25.4 25.8 29.7 29.4 30.3 32.6 32.2 31.7 33.4 32.3 38.5
Fish Liver Oil Mixtures 1.65 1.74 7.70 7.78 22.6 23.1 23.1 24.2 29.3 27.1 33.4 33 9 51.0 50.8 69.0 67.8
0,925 0.958 0.996 0.983
Hickman Distillates 3.57 3.60 25.9 25.0 28.1 27.8 131.5 133.4
1.009 0.065 0.982 1,014
1.055 1.010 1.022
1.045
ROUTIKE SPECTROPHOTOMETER AND
T-ITAUETER E:?m. VALUES. In Table X are included spectrophotometric and vitameter values determined in routine laboratory practice. These data demonstrate that reliable values can be obtained by making a minimum number of runs on each instrument. The corresponding values are in close agreement throughout, as is shown by the ratio values in the column a t the right. This again emphasizes the fact that the two instruments are capable of measuring the same characteristic of the fish liver oils, irrespective of their potencies or type. CONVERSIOX FACTOR. All seven oils studied in the long series on both spectrophotometer and vitameter were subjected to bioassay according to the U.S. P. procedure (9). Table XI gives these data as U. S. P. units per gram, and also the E::,n, values at 328 mp for both instruments. From these values one can calculate the so-called conversion factors for the different oils. I n the main these data show a remarkable agreement. One oil (No. 16,519) is 16 per cent lower than the mean from the spectrophotometer data and 15 per cent lower than the mean from vitameter data. Taking oils from the spectrophotometer, the mean conversion factor is 2152, but 3 per cent different from the factor calculated from the E:?m. value of the unsaponifiable fraction of the U. S.P. reference
INDUSTRIAL .4ND ENGINEERING CHEMISTRY
644
VOL. 12, NO. 11
B y observing these essential factors, a long series of tests was made on each of seven fish liver oils having a wide range in vitamin A potency. Analysis of the spectrophotometric Calculated Conversion E:?mm. Value a t 328 mF data shows that the mean of as fex as two deterFactor Bioassay SpectroSpectrominations gave a satisfactory result ( ~ per 2 T y p e of Fish Liver Oil value photometer Vitameter photometer Vitameter cent) . L’. s. P. unitslg I n the study with the vitameter, reproducibility was determined by making a series of tests 2066 2019 31.46 32.20 Halibut (3898) 65,000 1806 1796 60.39 Halibut (16,519) 50,500 50.12 on the same fish liver oils mentioned in connec2355 2321 78.68 79.85 Mixed (4288) 185,300 2295 2225 hlixed (16,319) 295,100 128.60 132.60 tion with the spectrophotometer. Analysis of Halibut (18,869) 31,900 15 05 15.12 2120 2110 the data established that a satisfactory 2191 40 07 2203 hlixed (16,949) 39.85 87,800 2222 2150 1.37 Cod “nonsap.” (9758) 1.35 3,000 value can be obtained from a minimum number Mean value 2152 2122 of six tests. The vitameter mean values on the lone: series were in close agreement with those of t h i spectrophotometer. ” By correlating the spectrophotometer and vitameter data oil, 2222. For the vitameter, the mean conversion factor is with those from the biological procedure, conversion factors 2122, also but 3 per cent different from that derived from the have been calculated. The several values are in good agreeunsaponifiable fraction of the reference liver oil, 2190. The ment, the mean value being, in this instance, 2137. This is, average of the mean conversion factors in Table XI, 2137, is therefore, a close approximation between the potency of a in close agreement with that obtained from routine bioassay fish liver oil in U. S. P. units per gram and the spectrophotoand spectrophotometric results on fish liver oils during the past several years. It is also very close to Barthen’s factor metric Et?m. value. 2064 referred to by Wilkie (IO). It is thus apparent that i t is possible, under the conditions of Conclusion technique as carried out (1) to obtain a quantitative evaluaThe E: value can be accurately determined spectrotion of vitamin A by measuring the extinction coefficient a t phot,ometrically under a carefully controlled procedure. It 328mp; and (2) by applying a conversion factor such as 2137, is therefore possible, b y employing the proper conversion to express the potency in U. S. P. or International units per factor, to evaluate satisfactorily the vitamin A potency of gram. fish liver oils in units per gram. Discussion and Summary TABLE XI.
CORRELATION OF E:?m. AT 328 ~p WITH BIOLOGICAL POTENCIES OF FISH LIVEROILS
Fm,
I n this critical study of the applicability of the spectrophotometric method to the determination of vitamin A in fish liver oils, the characteristics of the operative procedures have been carefully investigated with respect to the Bausch & Lomb spectrophotometer and the more specialized instrument, the Hilger vitameter. By means of an elaborate series of determinations on several oils, reproducibility has been demonstrated mith both, and a comparison of the results shows good agreement. The investigation has established the follov ing fundamentals with respect to the spectrophotometric technique: Since vitamin A in solution may lose considerable of its absorptive power after a time, the test reading should preferably be made within an hour after preparation of the solution. The vitamin is equally absorptive in isopropyl and absolute ethyl alcohols. The cells should be carefully paired and their surfaces kept scruDulouslv clean. EsDecial care should be taken that no oil residues remain on the cell surfaces. The values of a fish liver oil determined through a range of 50 times difference in concentration were found to be in agreement, showing that Beer’s law holds. This is also true when one concentration is examined in cells of different length, showing that Lambert’s law holds within the experimental error of the method. If a condensed spark is used as source of light, its position with respect to the optical axis of the system must be maintained with the highest degree of precision. This is ordinarily very difficult to accomplish since erosion of the electrodes is unavoidable during the course of sparking. On the other hand, if a Hilger hydrogen tube is employed, its constancy is definite -with respect to the optical axis, and one is able to obtain satisfactory results in regard to reproducibility and applicability to routine assays. For visual com arison, optimum results are obtained if the photographic emufsion has been processed to a density of 0.6. A few substances may interfere with t,he determination of vitamin A in fish liver oils. Among these the most important are: (a)the unsaturated long-chain fatty acids (particularly in lorn potency oils) and ( b ) the metallic salts such as copper oleate and ferric oleate, both of -which can be detected analytically if present in amounts having significant absorption.
Literature Cited (1) Carr, F. H., and Price, E. -I., Biochem. J . , 20, 497 (192G). (2) Drummond, J. C., and Coward, K. H., Ibid., 14, 734 ( 1 9 2 0 ) . (3) Drummond, J. C., and Morton, R. A., I b i d . , 23, 785 (1929). (4) Morton, R. A . , “Absorption Spectra of Vitamins and Hormones”, London, Adam Hilger, 1935. R. A,, and Heilbron, I. 31., Biochem. J . , 22, 087 (1928).
(5) Morton,
(6) Munsell,
H. E., “The Vitamins, A Symposium”, Chap. I V , Chicago, American Medical Association, 1938.
pp. 87-109,
(7) Rosenheim, Otto, and Drummond, J. C., Biochem. J., 19, 7 5 3 (1925).
(8) Takahashi, K., Kakamiya, Z., Kamakami, K., and Kitssato, T., Inst. Phys. Chem. Res. (Tokyo), Sci. Papers, 3 , 81 ( 1 9 2 6 ) . (9) U. S.Pharmacopeia XI (Second Supplement), p. 1 3 4 , 1939. (10) Wilkie, J. B., J . Assoc. Oficial Agr. Chem., 23, 336 ( 1 9 4 0 ) . PREBESTED before the Division of Biological Chemistry a t the 9Sth Meeting of the American Chemical Society, Boston, .\Iass.
T e m p e r a t u r e C o r r e c t i o n in F o r m u l a for V i s c o s i t y of L a t e x Is THE authors’ paper entitled “Examination of Rubber Latex and Rubber Latex Compounds. I. Physical Testing hIethods” [IND.ENG. CHEY., Anal. Ed., 9, 182-9 (1937)], Formulas 7 and 8 on page 188 should read 72.5 7’26
= 711 - 0.02 (25 - T)] = ~ ’ [-l 0.02 (25 - T)]
(7) (8)
These corrected formulas agree with the Crude Rubber Committee “Tentative Procedures for Testing the Variability of Xormal and Concentrated Latex” [ I N D . ENG. CHEX., Anal. Ed., 11, 593 (1939)l. The authors’ attention has been drawn t o this error in a private communication from W. S. Davey of the Rubber Research Institute of Malaya. H. F. JORDAX, P. D. BRASS,AND c. P. ROE
