Spectrographic Characteristics of Vitamin A Materials RONALD L. RIcFARLAN, PHILIP K . BATES, AND EDWARD C. MERRILL United Drug Company, Boston, JIass.
S
(2, 6 ) . Extraneous absorption occurs throughout the entire range of the absorption band. I n Figure 2 are shorvn the 11-hole oil and unsaponifiable fraction curves of a sample of U. S.P. reference cod liver oil within one month of the distribution date. The dip occurring a t the maximurn of the principal absorption band of the whole oil curve appears to exceed experimental error, and has been observed in fish liver oils before ( 1 ) . Also, a double secondary absorption peak occurs in the neighborhood of 2800, and was found on all four curves of which the curve in Figure 2 is the average. The maximum of the unsaponified fraction of the reference oil is very flat over the wave-length range 3225 t o
PECTROGRAPHIC methods have long been used for the quantitative estimation of vitamin A potencies. Although the fundamental soundness of the method is well established, it has been difficult t o obtain uniform interpret'ation of results when samples are evaluated in a large group of laboratories, using either a spectrograph or some one of the specialized devices employing the spectrographic principle. This paper reports some of the results of a recent study of the ultraviolet absorption of vitamin A with apparatus that permits an accuracy of detail not usually reported.
Procedure and Sources of Error All spectra used in the present experiments were obtained r i t h a Littrow gpectrograph havingoa linear dispersion of approximately 5 -4.per mm. a t 3280 A. A Hilger Spekker ultraviolet photometer was mounted in front of the spectrograph, and Hilger type J absorption cells for liquids-1 cm. in thickness-were mounted in the photometer. A condensed spark between tungsten steel electrodes was used as a light source. In order t o facilitate the accurate alignment of the electrodes, they were mounted on a de Gramont sparking stand instead of the usual holder supplied 11-ith Hilger Spekker photometer. Inaccurate alignment of the electrodes xas found to introduce an error in the drum reading of the Spekker photometer. Eastman type IV-0 plates were used, with exposure times varying from 5 to 40 seconds, depending on the concentration and also the portion of the absorption curve under investigation. A11 plates v;ere brushed during development. Line intensities were read by means of a Zeiss spectrum line photometer, and tests made on the reproducibility of intensity readings from a single line indicated that negligible error was introduced into the results by the microphotometer. The zero error in the drum reading of the Spekker photometer ii-as separately measured for each plate, and this error applied to the match-point determinations. All match points were determined by the spectrum line photometer by interpolating betneen the nearest microphotometer readings on either side of the match point. Over the short range of line densities involved near the match point the characteristic curve of the plate \vas assumed to be linear. The principal source of error in determining the match point is probably small variations in the sensitivity of the plate from one part of the plate to another. All samples mere prepared using isopropyl alcohol as a solvent, since little difference m-as found in curve shapes or peak positions using cyclohexane or isopropyl alcohol as solvents. The apparatus and procedure were checked by measuring the poiassium chromate absorption curve over the range 2900 to 3500 A. The curve obtained agreed with the expected values within the limits of experimental error (6). Comparative absorption spectra taken on a Hilger E-1 spectrograph ( in another laboratory) were found to yield identical curves to those taken on the above apparatus within experimental error.
/ 60
1.90 6%
-u- / . 2 0 Inn
I
1
1
I
I
I
I
28
ZS
30
31
32
33
28
29
I
I
1
1.40
1.20
& io0
-W
0.80
060
040
1
0.20 27
I
I
39
35
36
37
U. S. P. Reference Cod Liver Oil Measurements were made on a sample of U. S. P. reference cod liver oil during the period of 4 to 6 mont,hs after distribution. The absorption curves obtained are shown in Figure 1. Two sets of maxima are observed for the whole oil curves, the intensities of which change with increasing age of the oil. There is gradual decrease in the absorption a t both peaks, the absorption of the main peak decreasing with increasing age more rapidly than the absorption of the secondary peak. The maximum of the principal a b s o r p t i y band lies somewhat on the short wave-length side of 3200 A., while maximum of the unsap2nified fraction lies in the wave-length range 3225 to 3275 A. The extinction coefficients, are lower than would be expected for a fresh U. S. P. reference cod liver oil
27
30
31
32
33
34
WAVELENGTH. I O O A .
FIGERE 1. ABSORPTION CURVES 645
35
36
37
INDUSTRIAL h h D ENGISEERIKG CHEMISTRY
646
VOL. 12, NO. 1 I
foul different concentrations were made on a fresh sample of the U. S. P. reference cod liver oil. Table I1 shows the at several different wave lengths obtained a t values of these concentrations. Within the limits of experimental error E: appears constant and independent of concentration a t each of the several wave lengths listed. Accordingly, Beer’s lawoappears valid over the wave-length range 3100 to 3500 A. The average a t 3276 is close to the accepted value of 1.70 for the whole oil. The absence of several values for ci-because the match-point values of log & / A lie outside the range covered in the plate-appears not to affect the average, since obtained a t concentration c1 lie very close the values of E: to the average values for the various wave lengths.
5m.
K.
WAVELENGTH.
m a
FIGURE2 3275 1. Over this range the extinction coefficient,
for the unsaponifiable fraction is found to lie between 1.38 and 1.39, in good agreement with the currently accepted value (1. 3, 6).
Miscellaneous Vitamin A Materials In Figure 3 is shown the absorption curve of a commercial cod liver oil concentrate, which was manufactured by a saponification process. The principal absorption band is similar t o that of the unsaponifiable fraction of the U. S. P. reference oil shown in Figure 2; The presence of the first of the double peaks around 2800 A. may indicate either incomplete saponification or the presence of cod liver oil used for dilution to potency. Measurements taken on a sample of commercial Norwegian cod liver oil show the general characteristics of the referencegil curve in Figure 2, including the double peak around 2800 -4. 0 80 0
i
70
9 2 060 a
5 050
27
29
28
31
30
33
32
34
35
36
37
WAVELENGTH. 100 A.
FIGURE4
The results obtained from measurements made on the unsaponifiable fraction of the fresh reference oil are given in Table 111. In this series of experiments a single unsaponifiable fraction was obtained, and the concentrations were made by dilution of the original unsaponifiable fraction. About 30 minutes were required to expose completely a single plate during which time the solution containing the unsaponifiable fraction was left standing on the laboratory bench. The slow but consistent falling off in the absorption a t the various wave lengths seems to indicate a deterioration of the vitamin con-
040 COEFFICIENTS O F A L C O H O L A N D TABLEI. ABSORPTION ESTERF O R M S OF DISTILLED VITAMIN A ESTERS
030
A
.-Log
0 20 27
28
29
30
31
32
33
34
35
36
Wave Length
37
3475 3409 3343 3276 3258 3132
FIGURE 3
Beer’s Law at Various Wave Lengths In order to test the validity of Beer’s lam for various wave lengths of the absorption band, absorption measurements at
A
+ Concentration-
Alcohol
Ester
46.7 67.5 79.8 93.1 96.7 86.5
51.3 60.4
A.
WAVELENGTH, I O O A .
I n Figure 4 is shown the absorption curve of distilled vitamin A esters. Within experimental error the shape of this curve is similar to that of the unsaponifiable fraction of the U. S. P. reference oil shown in Figure 2. The saponification of the distilled vitamin A esters yielded the same absorption coefficients a t various wave lengths as the original distilled esters. Some of the values obtained are shown in Table I.
D-
82.6
90.6 91.1
82.7
TABLE11. BEER’SL A W AT \7AHIOUS W A V E LENGTHS, REFEREKCE CODLIVEROIL E170 1 cm.
Wave
Length
Cl
c2
1.05 1.31 1.51
0.95
ca
C1
u.8. P. Av
A. 3475 3409 3343 3276 3258 3132
..
.. ..
1.21 1.40 1.56 1.57 1.51
1.12 1.37 1.53 1.70 1.73 1.66
1.09 1.35 1.56 1.79 1.82 1.79
1.05 1.31 1.50 1.68 1.71 1.65
AShLYTICAL EDITIOK
NOVEMBER 15, 1940
T.IBLE 111. BEER'S LAM- &T L7ARIOES MT.iVE LESGTHS, ENs.4PONIFIABLE FRACTIOS OF 9. P. REFERENCE ( ' O D LIVER OIL
r.
Wave Length
-
E:%,. C1
C?
ca
CI
XV.
0.86 1.12 1.29
0.84 1.07 1.23 1.40 1.41
0.78 0.99 1.16 1.34 1.34 1.24
0.74 0.94 1.13
0 80 1.03 1.20 1.38 1.38 1.24
A. 3475 3409 3343 3276 3258 3132
1.46 1.47
1.2s
1.26
1.30 1.28 1.17
centration on standing for a few hours. The close agreement between the values $f E:?m. for t,he same concentration a t the wave lengths 3276 A. and 3258 A. indicates both the broadness of the peak and the precision of the procedure used. If allowance is made for the deterioration of the i-itamin on atanding, it Tvould appear that Beer's law holds for various r a v e lengths over the range measured.
Conclusions There appears to be a continuous change in absorption of the U. S. P. reference cod liver oil with use in the laboratory over an extended period of time, and as a standard for spectrographic measurements more rigid limitations are necessary than are now in use for biological assay. Fresh U. S. P. reference cod liver oil gives good agreement v ith the absorption values published by other experimenters.
647
Care must be exercised in using the spectrographic method to evaluate commercial cod liver oils and concentrates, because of the variability of the extraneous absorption. The distilled vitamin A esters show negligible change in their absorption properties upon saponification. Within experimental error the absorption curve shapes of the distilled vitamin A est,ers and the unsaponifiable fraction of the fresh U. S. P. reference oil are identical. Beer's law oappears to hold over the wave-length range 3100 to 3500 A. of the vitamin -4absorption curve.
-Acknowledgment The authors wish to express their appreciation for the assistance of David F. Rogers, who prepared samples for analyses, and to Distillation Products, Inc., which furnished the samples of vitamin A esters prepared by the molecular distillation of fish liver oil. Literature Cited S.,J. A m . Pharm. .Issoc., 26, 515 (1937). Holmes, A. D., et al., Ibid., 26, 537 (1937). H u m e , E. X I . , Nature, 143, 22 (1939). H u m e , E. M., a n d Chick, H., Medical Research Council, Brit., Special R e p t . Series 202, 30 (1935). International Critical Tables, Vol. 5 , p . 330, New York, McGrawHill Book Co., 1929. J I c F a r l a n e , W. D., a n d Sutherland, ..I. J., Can. ,J, Research, 16-B, 421 (1938).
I l j Bartlien, C. L., and Leonard, C .
(2) (3) (4)
(5) (6)
PRESENTED before the Division of Biological Chemistry a t the 99th lIeeting of the .lmericsn Cheniical Society. Cincinnati. Ohio.
The Measurement of Gloss L. A. WETLAUFER AND W. E. SCOT1 E. I. du Pont de h'emours & Co., Philadelphia, Peiina.
G
LOSS is a major component of the general appearance of an organic coating and often has direct bearing upon the serviceability of the film. Since visual observation of this property does not provide a n adequate means of recording exact reflection characteristics and, therefore, furnishes but a meager basis for comparing gloss observations which are made a t different periods, and since product standards, both wet and dry, may drift toward either higher 01 lower gloss on aging, an accurate method of measurement is important. No method for the measurement of this property appears to be in general use It is believed that insufficient attention to two important factors is largely responsible for the lack of instrumental application. First, and highly important, is the matter of eliminating extraneous factors, such as macroqcopic texture, permitting thereby a measurement of gloss per se. It must be recognized that any factors affecting appearance which are not inherent in the paint itself as manufactured and before it is laid down as a paint film-for example, factors which are introduced by the method of application-must be eliminated or controlled if one wishes t o ensure the uniformity of the product by physical testing. Secondly, instruments which have been offered commercially have, in the opinion of the authors, not provided the necesw y flexibility to enable satisfactory measuring of both high and low gloss or have been lacking in the precision required for adequate correlation with visual observations, Since the ultimate goal of the work herein described was to
provide a simple and yet sufficiently accuiate method for x-riting correct specifications and finally to establish a method which could be applied practically to the manufacturing operation, it seemed obvious that the conditions necessary for attainment of this goal could best be established through a fundamental study of the reflection characteristics of all types of surfaces common to the coating industry. The measurement of the spatial distribution of light reflected from a surface under given illumination is known as goniophotometry. I n practice, the reflection distribution is determined for a given angle of illumination in a plane normal to the surface. From such data it is then possible to select the angles of illumination and viewing, or possibly the one angle sufficient for the control of a given product, no matter \There it falls in the gloss range. Having thus explored the gloss range and having provided means for preparing films free from extraneous influences, it should be possible t o indicate a suitable method for routine control. Several investigators, notably Jones (6))Judd and Hunter (4, 5 ) , and Hanstock ( 3 ) , have made goniophotometric inrestigations of various surfaces, but there is no suitable instrument commercially available nor has there been published what appears to be a sound basis for the development of a satisfactory method for routine control work. This paper describes the work leading up to the final stagenamely, that of designing as simple an instrument as possible commensurate with the indicated requirements in precision
