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By M. J. Caldwell and D. B. Parrish (From The Kansas Agricultural Experiment Manhattan) (Received For Publication, December 26, 1944)
By M. J. Caldwell and D. B. Parrish (From The Kansas Agricultural Experiment Manhattan) (Received For Publication, December 26, 1944)
EFFECT
CARR-PRICE
OF LIGHT
ON THE STABILITY
OF THE
COLOR IN THE DETERMINATION
OF VITAMIN
A*
BY M.
(From
the Kansas
(Received
J. CALDWELL
AND
Agricultural
for
D.
B. PARRISH
Experiment
publication,
December
Station,
Manhattan)
26, 1944)
Procedure
The Coleman universal spectrophotometer,
model 11, with wave band
set at 620 rnp, was used in this study.
A resistance was placed in the
circuit of the exciter lamp so that the brilliance of the light could be
* Contribution
No.
298, Department
of Chemistry.
181
182
DETERMINATION
OF
VITAMIN
RESULTS
AND
DISCUSSION
varied without
dimming the light in the galvanometer
unit.
Matched
13 mm. square absorption cells were used. A vitamin A concentrate (Distillation Products, control, PC-2) was dissolved in U. S. P. chloroform and
diluted to approximately
5 y per ml. After the usual preliminary adjustments of the galvanometer were made, an absorption cell containing 1 ml.
of the vitamin A solution was placed in the light path and 9 ml. of 22.5
per cent antimony trichloride reagent (2) were added from a special pipette
(8). The first reading of the galvanometer was made as soon as temporary
stability was observed.
Although varying slightly, depending upon the
intensity of the exciter lamp, this point of stability occurred approximately
6 seconds after beginning the addition of the reagent.
Further observations of the galvanometer were made at the time intervals shown in Fig.
1, with other intermediate
intervals omitted from the figures to avoid
crowding.
The values shown are averages of several determinations.
The
intensity of the exciter lamp was adjusted to 13,30, and 100 per cent of the
full brilliance.
The observations marked 0 per cent were taken by shifting
the absorption cell containing the reaction mixture into the light beam
(at 13 per cent of full brilliance) only long enough to read the galvanometer;
generally this required about 3 seconds.
To study the effect of very strong illumination, the chloroform solution
of vitamin A and the reagent were mixed and exposed intermittently
to a
200 watt Mazda lamp at 3 inches distance.
The absorption cell containing
the reaction mixture was removed from the strong light only long enough to
transfer it to the photometer and read the galvanometer.
The same stock solution of vitamin A was used to study the loss of color
in the Evelyn and K W S Z photometers
with the 620 rnp and 625 rnp
filters respectively.
The K W S Z was adapted for rapid reading by
adjusting the setting on the decade box to the expected transmission.
The
reaction was carried out with the absorption cell in the photometer and any
necessary adjustment
of the transmission
was made by reading the small
galvanometer
deflection which had been previously
calibrated in terms
of the decade box readings.
M.
J.
CALDWELL
AND
D.
B.
183
PARRISH
following mixing of the reagent with vitamin A. The values shown for the
Coleman photometer were all obtained by using the calibration curve prepared with the incident light at 13 per cent normal brilliance.
The data in Fig. 1 and Table I show clearly that light exerts a powerful
effect in the fading of the blue color. As the incident light of the Coleman
photometer is changed from 100 to 13 per cent of full normal brilliance, the
rate of loss of color during the first minute decreases from 39 to 18.2 per
IO 20
30
40
50
TIME
60
IN
90
SECONDS
120
150
It30
Curvesl,2,
FIG. 1. Fading
of Carr-Price
color as a function
of light
intensity.
3, and 4 are obtained
with a Coleman
photometer,
when the reaction
mixture
is exposed continuously
to 0, 13, 30, and 100 per cent of the normal
photometer
light
intensity.
Curve
5 shows the effect of exposure
to direct illumination
from a 200 watt
Curves
6 and 7 represent
normal
fading obtained
when the Evelyn
incandescent
lamp.
and K W S Z photometers,
respectively,
are used.
cent. Exposure of the solution to light at 13 per cent of normal for only
long enough to read the galvanometer reduces this loss of color to 11.7 per
cent. The loss of color is 89.4 per cent if the light of a 200 watt bulb is
allowed to strike the solution.
A smaller loss, 7.8 per cent, is observed if
light of low intensity such as that employed in the Evelyn photometer is
used. The extrapolation
of all curves to zero time tends to bring all the
curves toward a common point.
As would be expected from a study of the
curves, there is a tendency for the photometric
reading to be lower when
184
DETERMINATION
OF
VITAMIN
TABLE
Rate
of
Fading of Carr-Price
Photometer
Color
I
from
Light
Initial
to 60 Second
intensity
Reading
Rate of color
loss
!-
Coleman.
,_..__.__.
KWSZ
__.__...._
Evelyn.
0 normal
Normal
lamp
89.4
39.0
27.6
18.2
11.7
32.6
7.8
through six pieces of glass and an aqueous solution before reaching the
absorption cells; and yet fading was very rapid.
Hock (6) studied the kinetics of the Carr-Price reaction, using a photographic device to record color density. Fig. 4, a of his paper shows that
vitamin A naphthoate fades so rapidly that if the same rate had continued
for 70 secondsthe value would have been practically 0 per cent of the maximum color developed. The results of this study indicate that a lessintense
light source would have causedlessrapid fading. In making kinetic studies
of the Carr-Price reaction, one might be led to assume either a zero or a
first order reaction, depending upon the intensity of light employed in the
photometer. Meunier and Raoul (9) studied the kinetics of the CarrPrice reaction of vitamins A1 and A,. The intensity of the light source
may causethe two forms of the vitamin to fade at different rates than those
found in their study and merits further investigation.
Since the determination of correction factors for the presence of carotenoids interfering
M.
J.
CALDWELL
AND
D.
B.
PARRISH
185
SUMMARY
1. The intensity of the incident light influences the rate of fading of the
blue color developed in the Carr-Price reaction for vitamin A.
2. Investigations
of the kinetics of the Carr-Price reaction should take
into account the effects of the illumination in the photometer.
3. Photometers
for determining vitamin A by the Carr-Price
reaction
should employ low intensity of incident light to reduce fading of the blue
color to a minimum and make possible more precise determinations.
BIBLIOGRAPHY
1. Dann,
2. Koehn,
186
3.
4.
5.
6.
7.
8.
9.
DETERMINATION
OF
VITAMIN
Norris,
E. R., and Church,
A. E., J. Biol. Chem., 86,477
(192930).
Almquist,
H. J., Mackinney,
G., and Mecchi,
E., J. Biol. Chem., 150,99
Notevarp,
O., and Weedon,
H. W., Biochem.
J., 30, 1705 (1936).
Hock,
H., Biochem.
J., 37, 425 (1943).
Urban,
F., Milder,
B., and Carruthers,
C., Biochem.
J., 37,295
(1943).
Parrish,
D. B., and Caldwell,
M. J., J. Lab. and Clin. Med., 29,992
(1944).
Meunier,
P., and Raoul,
Y., Compt.
rend. Acad., 206, 1148 (1938).
(1943).
ARTICLE:
THE EFFECT OF LIGHT ON THE
STABILITY OF THE CARR-PRICE
COLOR IN THE DETERMINATION OF
VITAMIN A