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- 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

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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. 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

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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

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

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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 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

Coleman. ,_..__.__.

KWSZ __.__...._ Evelyn.

-

Light intensity Rate of color loss

!- 200 watt Mazda lamp 100% normal 30y0 normal 1394 0 normal Normal

@2r cent per min.

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 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 seconds the value would have been practically 0 per cent of the maxi- mum color developed. The results of this study indicate that a less intense light source would have caused less rapid 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 cause the 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

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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 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).

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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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M. J. Caldwell and D. B. Parrish

VITAMIN ACOLOR IN THE DETERMINATION OFSTABILITY OF THE CARR-PRICE THE EFFECT OF LIGHT ON THEARTICLE:

1945, 158:181-186.J. Biol. Chem.

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