Simplified Form of Morton and Stubbs Correction of Vitamin A Absorption Curves BERNARD L . OSER Food Research Laboratories, Inc., Long I-sland City, N. Y . ECENTLY, Morton and Stubbs (1-3) proposed a mathematical means for correcting absorption spectra of fish liver oils for irrelevant absorption in the region of the vitamin A peak. The correction is made in two stages, the first correcting for distortion in the shape of the true vitamin A curve and the second correcting for the vertical displacement of the curve due to nonvitamin A absorption. The basic assumption upon which the first correction is predicated is that interfering nonvitamin A substances absorb linearly within a narrow region on either side of the vitamin .A absorption maximum-viz., between wave lengths a t which the absorption of pure vitamin A is 6/7 maximal. For a discussion of the geometric basis for the Morton and Stubbs correction, the reader is referred to the original papers. The present report describes a simplified procedure for computing the Morton and Stubbs correction. This will be found convenient in cases where it is not necessary to plot the corrected absorption curves or the curves for irrelevant absorption.
Table 1 .
Vitainin .i Solvent AUoohol Cyclohexane Ethanol Isopropyl alcohol Acetate Cyclohexane Ethanol Isopropylalcohol
mg 311 311 312 313 311.5 313
IC,
m ,m p 326 325 326 328 326.5 328
y, mp
336 335.5 336 338.5 337.5 338.5
-a ) 7a 7(1 - n ) 2.800 4.200 3.000 4.000 2.917 4.082 2.882 4.118 2.962 4.03s 2.882 4.118
lengths z and y below and above the peak, m, where absorption is 6/7maximal; a n d a = (y - r n ) / ( y - 2). When the peak absorption is a t 326 millimicrons and the 6 / 7 “fixation points” are 312 and 336 millimicrons (as determined i n the author’s laboratory for vitamin A alcohol in isopropyl alcohol) the numerical constants may be substituted in the general Equation 1:
H v combining the correction for slope s s =
\ d u e s of 7n and 5 (1
E32n(corrected) =
(Es- Ev)‘ 2
:E326
- 2.9173312
- 4.082Em
(2)
Y - X
When Ea2ais taken as unity the other E values beconie rstinction ratios. 111 that case this equation becomes
with the correction for height h
(E, - S) - h Ey* - h
-7 -g
f
in whichf is the factor by which the observed Ew, is multiplied t’o obtain the corrected value. The critical wave lengths for peak absorption and the fixation points depend upon the solvent employed (ethanol, cyclohexane, or isopropyl’ alcohol) and on whether the vitamin is in solution as free alcohol (unsaponifiable extract) or ester. Values for t,he coefficients 7a and 7(1 - a ) as calculated from the data of llorton and Stubbs (3) for cvclohexane and ethanolsolution~and from the author’s own data for isopropyl alcohol, are given in Table I. To facilitate the use of the equation, a nomogram may be constructed based on the observed values for rn, z, and y. An example is shown in Figure 1. By merely connecting the point,s corresponding to the calculated extinction ratios a t 312 and 336 millimicrons, one can read the correction factor, f , on the central axis. It is hoped that this simple device will facilitate more widespread trial of the Morton and Stubbs correction with a view toward determining its validity.
* or E,, whichever is lower the equation for the corrected extinction coefficient a t the absorption maximum, E,(corrected) = E,, - s - h becomes E,(corrected) = 7 E ,
- 7aEz - 7(1 -
a)E,
(1)
in which E,,, is the extinction coefficient at the absorption maximum for vitamin A; E, and Ey are the coefficients a t wave
f 0.3 0.4
0.95
1
0.5 0.6 0.7
o,90f
0.8 0.9 1.00 1.10
-~
0.854
_i
I
0.801
j j
-
0.15
0.70
7 - 2.917E311/326 - 4.082E336/326
Vitamin A alcohol in isopnopanol f = 7 - 2.917~ - 4.082 y E,,, (corpected) = fxE32s
Directions: Extend line between values Read f on center scale. Figure 1.
for,
LITERATURE CITED
(1)
Morton, R. A . , and Stubbs, A. L., Analyst, 71, 348
(2)
Morton, R. A., and Stubbs, A. L.. Biochem. J., 41, 525
(3)
Ibid., 42, 195 (1948).
(1946).
E 312/326 and E336,326,
Nomogram for Morton-Stubbs Correction
529
(1947). RECEIVEDSeptember 25, 1948
