J. Biol. Chem.-1946-Caldwell-565-72

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CHANGES IN THE ABSORPTION SPECTRA DUE TO AGING OF
THE CARR-PRICE REACTION MIXTURE WITH VITAMIN A
AND THE COMMON CAROTENOID PIGMENTS*
BY M. J. CALDWELLt AND J. S. HUGHES
(From the Kansas Agricultural Experiment Station, Manhattan)

(Received for publication, September 2, 1946)

Carr and Price (1) in 1926 introduced a reagent for the quantitative
estimation of vitamin A which was destined to play an important rble in
the study of the physiology of the vitamin. This reagent, a saturated

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solution of antimony trichloride in chloroform, still occupies a position of
first importance in most laboratories where vitamin A is estimated by
chemical means. The sensitivity of the reagent and the simplicity of its
application have made its use in many cases the method of choice. How-
ever, its non-specificity and the transient character of the blue coloration
produced with vitamin A have at times cast doubts on its general reliability.
Although numerous non-vitamin A substances are known to be chromo-
genic with antimony trichloride (2), difficulty is usually encountered only
in the case of the carotenoid pigments which are closely related to vitamin
A and which frequently accompany the vitamin in animal tissue. The
colors produced by these pigments with the reagent display distinctly dii-
ferent absorption spectra and a much greater apparent stability than that
due to vitamin A. The effect of increased temperature’ and of intense
illumination2 on the instability of the Carr-Price colors is much more pro-
nounced in the case of vitamin A than in the case of the carotenoids. The
variation in stability has been made the basis for the differentiation of the
vitamin A color from that of the other chromogens by several investigators
(3-5).
Several workers have reported constants relating to the absorption
spectra of the Carr-Price reaction products of vitamin A and of several of
the other chromogenic substances. Gillam (6) has reported the wave-
length of the absorption maxima and the corresponding E::,. values for p-
carotene, lycopene, lutein, zeaxanthin, and vitamin A. Goldhammer and
Kuen (7) have given corresponding values for carotene, xanthophyll, and
for several of the sterols. Von Euler et al. (8) have published curves show-
* Contribution No. 321, Department of Chemistry.
t From the dissertation submitted in partial fulfilment of the requirements for the
degree of Doctor of Philosophy in Chemistry from the Graduate School of Kansas
State College.
1 Caldwell, M. J., and Hughes, J. S., in preparation.
a Caldwell, M. J., and Hughes, J. S., in preparation.
565
566 CARR-PRICE RJSACTION PRODUCTS

ing absorption characteristics for g-carotene immediately after adding the
reagent and also 30 minutes later. Lamb, Mueller, and Beach (9) have
published curves showing the antimony trichloride colors with ergosterol,
cholesterol, and 7dehydrocholesterol. Gibson and Taylor (10) recently
introduced a new technique for observing the rapidly changing spectra of
the antimony trichloride-chromogen systems. Their “dynamic method”

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SPLCTNOPNOlOMElEN
Fra. 1. Modified form of the flowing cell apparatus of Gibson atnd Taylor used in
de :&mining the absorption spectra by the “dynamic method.”

involves the use of a “flowing cell” (Fig. 1) in which a steady state of flow-
ing reagent and chromogen is maintained during the spectral measurement.
These workers have presented curves for liver oil concentrates, their oxi-
dized products, and for p-carotene, which show the rapid changes as’the
solution ages.
No comprehensive investigation has been found in the literature relating
to the changing spectra of the antimony trichloride reaction products with
vitamin A in its various forms and with the carotenoid pigments most likely
M. J. CALDWELL AND J. S. HUGHES 567

to interfere with the Carr-Price determination of vitamin A. It is the
purpose of this paper to present these fundamental data which should prove
of value in the further development of the Carr-Price method.
Procedure
Materials-The Carr-Price reagent was prepared by the general method
of Koehn and Sherman (11). The antimony trichloride was dissolved in
the purified chloroform at the rate of 22.5 gm. per 100 ml. of chloroform.
The reagent was stored at room temperature in darkness or subdued light
and filtered before use. Diierent batches of reagent exhibited great
uniformity and stability.
Vitamin A3 as the crystalline alcohol, crystalline acetate, and the liquid

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concentrate of the natural esters was available for the investigation. These
preparations were preserved in the dark at -20” until ready for use.
Stock solutions in U. S. P. chloroform were prepared and from these suitable
dilutions ranging from 5 to 50 y per ml. were made for use in the Carr-Price
study.
The seven most common carotenoid pigments* listed by Zechmeister (12)
were available for these studies. These were a-carotene, /3-carotene,
y-carotene, lycopene, cryptoxanthin, lutein, and zeaxanthin. Although
the quantity of y-carotene available was insufficient for the study of its
Carr-Price absorption spectrum, it was used in the other phases of the work
to be reported. These pigments were also stored in the dark at -20” until
used. Solutions in U. S. P. chloroform ranging in concentration from 100 to
400 y per ml. were prepared, and the concentration and purity established
in all cases by reference to the spectral data. to be found in the literature
(12, 13). The absorption data were obtained by the use of the Beckman
spectrophotometer.
Apparatus and Methods-Most of the data here reported were obtained
by use of the Beckman spectrophotometer. Before the spectral measure-
ments were made the wave-length dial was set at the desired point and the
instrument adjusted to zero optical density with a “blank” mixture of 1 ml.
of chloroform and 9 ml. of the reagent. 1 ml. of the solution under test
was placed in a lipped tube and 9 ml. of the reagent added. At this instant
a stop-watch was started. The reaction mixture was transferred to the
8 The vitamin A preparations were generously supplied by Dr. P. L. Harris of the
Distillation Products, Inc.
4 Dr. L. Zechmeister of the California Institute of Technology contributed samples
of lycopene and zeaxanthin which were used in this study. Dr. John Porter of Purdue
University and Dr. G. Mackinney of the University of California kindly supplied the
htein and the cryptoxanthin, respectively. The remaining carotenoid pigments
were either prepared in this laboratory or purchased.
568 CARR-PRICE REACTION PRODUCTS

absorption cell, which was quickly placed in position for measurement in
the Beckman spectrophotometer. At 30 seconds the first reading of
optical density was taken, and readings were taken at intervals thereafter
for a period of 10 minutes. This set of data represented the change in the
optical density at the chosen wave-length, with time. The wave-length
dial was reset, and the measurements repeated. This process was repeated
at frequent intervals in the wave-length range from 500 to 700 mp, well on
either side of the 620 m/l maximum of the vitamin A Carr-Price reaction
product. In plotting the data, all of the optical densities obtained at the
same time after mixing were plotted against the wave-length. A series of
absorption curves was thereby obtained, each member of which corresponds
to a specific age of solution. These curves, viewed together, present a

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picture of the changing Carr-Price reaction product from 30 seconds to
10 minutes after mixing.
For time intervals less than 30 seconds recourse was had to the “dynamic
method” of Gibson and Taylor, with a modified flowing cell apparatus.
Fig. 1 shows the essential features of the flowing cell which was placed over
the aperture of a visual Bausch and Lomb spectrophotometer. The
reagent and chromogen solutions were allowed to mix at a constant rate
and flow through the absorption cell at a time after mixing controlled by
the level held in the retention chamber. When a steady state was reached,
the absorption curve of the flowing mixture was determined in a normal
fashion. The time after mixirg was calculated by dividing the ml. of
solution between the point of mixing and the center of the observation
window by the rate of flow of the solution in ml. per minute. With the
apparatus used, reproducible results could be obtained with mixtures from
2 to 30 seconds of age.
Calculations-All optical densities were converted to the corresponding
E:Fmm.values by means of the Bouger-Beer (Beer-Lambert) law. Thus

where D is the observed optical density, c is the concentration in per cent of
the chromogen, and 1 is the thickness of the absorption cell in centimeters.
DISCUSSION

The results obtained in this investigation are presented graphically in
Figs. 2, 3, and 4. In these figures the E:‘?&. values are shown plotted
against the wave-length, which extends well on either side of the 620 rncc
maximum of the vitamin A Carr-Price reaction mixture. The age of the
solutions measured from the instant of mixing is indicated on the various
curves by numbers as well as by the coded lines. The solid lines represent
M. J. CALDWELU AND J. S. H.UGHES 569

the solution of least age, while the dotted lines represent the oldest of the
solutions.
Examination of the curves reveals marked differences between those re-
lating to vitamin A in any of its forms and the curves of the carotenoid
pigments. Vitamin A is seen to have chromogenic powers lo- to 25-fold
500
4 VITAMIN A ALCOHOL k------O ‘EC’

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660 700

VITANIR A ACETATE -----306EC

----L MIN.

-- 6141~.

500 560 600 650 100

--V,TAYIN A
CONCENTRATE

500 660 600 660 700
WAVE LENGTH IN Mll

FIG. 2. Absorption spectra of the Carr-Price reaction products with vitamin A as
the alcohol, acetate, and the concentrate of the natural esters, at various times after
mixing. The E (l’%, 1 cm.) values are calculated in terms of the alcohol equivalent.

greater than the common carotenoids in the region of the vitamin A
maxima. Vit,amin A, as alcohol, acetate, or natural ester, is unique among
the chromogens in possessing a single strong absorption band (maximum at
620 rnp) which rapidly decreases with time. Changes of a more complex
nature are exhibited by p-carotene, lutein, and zeaxanthin. Here the
absorption maxima shift as a wave or in steps toward the red end of the
spectrum as the solution ages. The remaining chromogens, a-carotene,
570 CARR-PRICE REACTION PRODUCTS

lycopene, and cryptoxanthin, show relatively simple absorption spectra,
which gradually rise or fall with time. In no case can the Carr-Price color
be termed %table.”
A study of these absorption curves makes clear the differences in the
“stability” of the Carr-Price colors, as normally measured with a filter
calorimeter or a diffraction instrument of equally wide wave band. In the

-
800 SET1 EWE
CAR01
$400
$;;mmme &+.4$
100-

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- -eslIc.
0
500’ 550 600 650 7bO

so0 ----II HOURS
LUTEIN r ---IO
c.-- t I MIN.
g400

“--A+g
w
--30 SEC.
1 L-----es,,.
01 * ’ ’ ’ ’ ’ ’ ’
so0 510 600 SSO 700

I HOUR
r-
*- I rSMlN.

01 ’ ’ ’ ’ ’ ’ * ’
500 350 000 610 100
WAVE LENGTH IN “)I

FIG. 3. Absorption spectra of the Carr-Price reaction prodkts with &carotene,
lutein, and zeaxanthin at various times after mixing of the reactants.

case of vitamin A, only the decreasing absorption in the region of 620 rnp
is recorded, while in the case of the carotenoids major changes occurring
in the character of the absorption spectra may be entirely overlooked, due
to the width of the filter band used in making the measurement. Thus,
the Carr-Price colors with the various carotenoid pigments may be re-
corded as stable, increasing, or decreasing, depending on the integrated
area under the absorption curves between the limits imposed by the
optical system involved in the measurement.
M. J. CALDWELL AND J. S. HUGRES 571

SUMMARY

1. Spectral absorption curves for the Carr-Price reaction mixture with
vitamin A’as alcohol, acetate, and concentrate of the natural esters have
been presented for reaction mixtures of ages varying from 2 seconds to 10
minutes.

ALPHA CAROTENE

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500 550 600 SW 700

5oo- LYCOPENE

01 . I I I I I * 1
500 550 600 650 700

01 * 1 0 ’ 3 ’ n ’
500 550 600 650 700
WAVE LENOTH IN MJl

FIG. 4. Absorption spectra of the Carr-Price reaction products with a-carotene,
lyeopene, and cryptoxanthin at various times after mixing of the reactants.

2. Similar data have been presented for six of the common carotenoid
pigments, a-carotene, B-carotene, lycopene, cryptoxanthin, lutein, and
seaxanthin.

The authors wish to thank Dr. L. Zechmeister, Dr. John Porter, Dr. G.
Mackinney, and Dr. P. L. Harris for the gift of the carotenoids and the
vitamin A preparations used in this study. Further, we wish to acknowl-
edge the technical assistance of Mrs. Howard Hamlin who aided materi-
ally in this project.
572 CARR-PRICE REACTION PRODUCTS

BIBLIOGRAPHY

1. Carr, F. H., and Price, E. A., Biochem. J., 20,497 (1926).
2. Delaby, R., Sabetay, S., and Janot, M., Compt. rend. Acad., 198,276 (l934).
3. Oser, B. L., Melnick, D., and Pader, M., Ind. and Eng. Chem., Anal. Ed., 16,724
(1943).
4. Brew, W., and Scott, M. B., Ind. and Eng. Chem., Anal. Ed., 18,46 (1946).
5. Meunier, P., and Raoul, Y., Compt. rend. Acad., 208,1148 (1938).
6. Gillam, A. E., Biochem. J., 29, 1331 (1935).
7. Goldhammer, H., and Kuen, F. M., Biochem. Z., 967,406 (1934).
8. von Euler, B., von Euler, H., and Hellstrom, H., Biochem. Z., 203, 370 (1928).
9. Lamb, F. W., Mueller, A., and Beach, G. W., Ind. and Eng. Chem., Anal. Ed., 18,
187 (1946).
10. Gibson, G. P., and Taylor, R. J., Analvat, 70, 449 (1945).
11. Koehn, C. J., and Sherman, W. C., J. Biol. Chem., 132,527 (1940).

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12. Zechmeister, L., Chem. Rev., 34, 267 (1944).
13. Zscheile, F. P., White, J. W., Jr., Beadle, B. W., and Roach, J. R., Plant Physiot.,
17,331 (1942).