THE EFFECT OF LIGHT ON THE STABILITY OF THE

CARR-PRICE COLOR IN THE DETERMINATION

OF VITAMIN A*
BY M. J. CALDWELL AND D. B. PARRISH
(From the Kansas Agricultural Experiment Station, Manhattan)

(Received for publication, December 26, 1944)

The Carr-Price reagent, a chloroform solution of antimony trichloride,

has been widely used in the determination of vitamin A. The blue color
developed upon mixing vitamin A and the reagent is unstable; fading be-

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gins immediately. The attempt to make photometric readings which are
reproducible has led to a variety of practices in different laboratories. For
example, galvanometer readings have been made at 3, 5, 15, 30, and 60
seconds after adding the reagent; the point of tempora,ry stability has been
used; curves have been plotted and extrapolated to zero time, while still
others have employed photometers making use of condensers and ballistic
galvanometers or photographic devices for measuring t,he color density.
Some investigators have called attention to the difficulty of determining
the point of temporary stability, although others have not found that this
necessitates special consideration (l-7).
In this laboratory several different photometers have been available for
determining vitamin A by the Carr-Price reaction. It has been observed
that the stability of the blue color varied considerably when different
instruments were used in the analysis. Since intensity of the light source
appeared to cause the difference, this study was undertaken. Examination
of the literature failed to reveal studies of the effect of light upon the Carr-
Price reaction mixture.
These observations should be of interest to those using this method for
the determination of vitamin 9. The results should lead to a better
underst,anding of the kinetics of the react.ion, show why differences in the
stability are often observed, contribute to a more exact determination of
correction factors for the presence of carotenoids in the reaction mixture,
and suggest a few factors to consider in the design of photometers for de-
termining vitamin ,4 by t.he Carr-Price method.

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 A

varied without dimming the light in the galvanometer unit. Matched

13 mm. square absorption cells were used. A vitamin A concentrate (Distil-
lation Products, control, PC-2) was dissolved in U. S. P. chloroform and
diluted to approximately 5 y per ml. After the usual preliminary adjust-
ments 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 observa-
tions of the galvanometer were made at the time intervals shown in Fig.

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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.

RESULTS AND DISCUSSION

The three photometers do not show identical galvanometer readings for

the same vitamin A solution because of differences in construction and size
of absorption cells. Therefore, to bring the results together and make
comparisons as clear as possible, all readings have been transposed to appar-
ent vitamin A concentration, by reference to the calibration curves
previously prepared for each photometer based on the color obtained at the
point of temporary stability. It should be noted that the curves in Fig.
1 also represent the relative color of the solution as it varies with time
 M. J. CALDWELL AND D. B. PARRISH 183

following mixing of the reagent with vitamin A. The values shown for the
Coleman photometer were all obtained by using the calibration curve pre-
pared 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

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0 IO 20 30 40 50 60 90 120 150 It30

TIME IN SECONDS

FIG. 1. Fading of Carr-Price color as a function of light intensity. Curvesl,2,

3, and 4 are obtained with a Coleman photometer, when the reaction mixture is ex-
posed continuously to 0, 13, 30, and 100 per cent of the normal photometer light in-
tensity. Curve 5 shows the effect of exposure to direct illumination from a 200 watt
incandescent lamp. Curves 6 and 7 represent normal fading obtained when the Evelyn
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 A

brilliant illuminations are used. The fading of a solution containing 10 y

of vitamin A per ml. also was studied, with results essentially the same as
shown in Fig. 1. The use of a perforated diaphragm to control the light
intensity was tried, with results essentially the same as those obtained
with rheostat control. Because of mechanical difficulties, the control
of the light system by rheostats was adopted.
Although light n-ill destroy vitamin A over a period of time, it was found
that exposure of the vitamin A solution to the 200 watt light at 3 inches
distance for 2 minutes before adding the antimony trichloride resulted in a
blue color as high as that found for non-irradiated vitamin A. The light
affects the blue color combination much more rapidly than it affects vitamin
A it,self. The fading of the blue color appears to be due to radiations of the

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red region of the spectrum, Ultraviolet light could have little if any effect
in this case, since in one of the photometers used the incident light passed

TABLE I
Rate of Fading of Carr-Price Color from Initial to 60 Second Reading
Photometer Light intensity Rate of color loss

200 watt Mazda lamp

!-
@2r cent per min.
89.4
Coleman. 100% normal 39.0
,_..__.__. 30y0 normal 27.6
1394 18.2
0 normal 11.7
KWSZ __.__...._ Normal 32.6
Evelyn. 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 photo-
graphic 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 maxi-
mum 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 Carr-
Price 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 deter-
mination of correction factors for the presence of carotenoids interfering
 M. J. CALDWELL AND D. B. PARRISH 185

with the determination of vitamin A may be closely related to the kinetics

of the reaction, this problem might profitably be reexamined, taking into
account the effect of light upon the reaction mixture.
Dann and Evelyn (1) show that fresh and old solutions of antimony tri-
chloride reagent cause fading to begin at different lengths of time after
adding the reagent to vitamin A. An investigator using a photometer of
different light int,ensity might erroneously conclude his reagent to be more
or less stable than those prepared by Dann and Evelyn unless the effect of
light is taken into account. The Evelyn photometer employs a minimal
light source.
A study of these data indicates why some investigators find that observa-
tions made at the point of temporary stability are reliable, while others do

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not. With low light intensity the drift is so slow that one may obtain
satisfactory reading over a period of several seconds, but with greater
illumination the time at which the reading should be made is much more
difficult to determine.
Measurements of vitamin A may be improved by using light of low
intensity. It \vas found that satisfactory measurements of vitamin A
could be made with the Coleman photometer when the intensity of the
exciter lamp was decreased to less than 10 per cent of full brilliance. Under
these conditions the calibration curve prepared approached a straight line,
just as was the case when the instrument was used in the normal fashion.
This method of adjusting the photometer has been adopted in this labora-
tory for the routine determination of vitamin A. However, calibration
curves should be prepared for the light intensity to be used in subsequent
determinations, since small differences in the readings are observed if the
light intensity is varied from low to high illumination. This is probably
due to slight differences in t,he character of the incident light and to changes
in the rate of fading.

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, W. J., and Evelyn, K. A., Biochem. J., 32, 1008 (1938).
2. Koehn, C. J., and Sherman, W. C., J. Biol. Chem., 132, 527 (1940).
186 DETERMINATION OF VITAMIN A

3. Norris, E. R., and Church, A. E., J. Biol. Chem., 86,477 (192930).

4. Almquist, H. J., Mackinney, G., and Mecchi, E., J. Biol. Chem., 150,99 (1943).
5. Notevarp, O., and Weedon, H. W., Biochem. J., 30, 1705 (1936).
6. Hock, H., Biochem. J., 37, 425 (1943).
7. Urban, F., Milder, B., and Carruthers, C., Biochem. J., 37,295 (1943).
8. Parrish, D. B., and Caldwell, M. J., J. Lab. and Clin. Med., 29,992 (1944).
9. Meunier, P., and Raoul, Y., Compt. rend. Acad., 206, 1148 (1938).

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 THE EFFECT OF LIGHT ON THE
STABILITY OF THE CARR-PRICE
COLOR IN THE DETERMINATION OF
VITAMIN A
M. J. Caldwell and D. B. Parrish
J. Biol. Chem. 1945, 158:181-186.

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