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[24] Colorimetric estimation of vitamin A with trichloroacetic acid
[24]
C O L O R I M E T R I C E S T I M A T I O N OF V I T A M I N A W I T H T C A
[2 4] C o l o r i m e t r i c
189
Estimation of Vitamin A with Trichloroacetic Acid
By R. F. BAYFIELD and E. R. COLE It is now over 50 years since the color reaction of vitamin A with strong acids, notably sulfuric acid, was first observed. 1'2 The importance of this test lies in the fact that it provided the basis for a general view of the reaction of vitamin A with Lewis acids, including the Carr-Price antimony trichloride reaction,3 which has tended to dominate colorimetric methods of estimation from that time. However, several unsatisfactory features of the Carr-Price method, particularly interference by moisture and instability of the color, have prompted continued investigations for alternative reagents. Of these, glycerol dichlorohydrin, 4 trifluoroacetic acidp and trichloroacetic acid 6 have received most prominence, but development of methods has proceeded in an empirical manner and only comparatively recently have mechanistic aspects of these reactions received consideration. It is in this context that the trichloroacetic acid method is now discussed with particular reference to estimation of vitamin A in serum and liver tissue. Experimental
Reagents. All reagents should be freshly prepared. Exposure of samples and reagents to light must be avoided at all times. Trichloroacetic acid, saturated solution in chloroform. Trichloroacetic acid crystals (50 g), freshly washed with solvent, are dissolved in alcohol-flee distilled chloroform (25 ml). This gives about 50 ml of reagent which is stable for 5 hr with daylight excluded. Optical density readings tend to decrease with storage for longer periods, up to 24 hr. No color is given by vitamin A with reagent exposed to bright diffuse sunlight for several hours. Vitamin A (U.S.P. Reference capsule vitamin A acetate), strong solu-
1 j. C. D r u m m o n d and A. F. Watson, Analyst 47, 341 (1922). 2 0 . Rosenheim and J. C. Drummond, Biochem. J. 19, 753 (1925). '~ F. H. Carr and E. A. Price, Biochem. J. 20, 497 (1926). 4 A. E. Sobel and H. Werbin, J. Biol. Chem. 159, 681 (1945);Ind. Eng. Chem. Anal. Ed. 18, 570 (1946). 5 R. E. Dugan, N. A. Frigerio, and J. M. Siebert, Anal. Chem. 36, 114 (1964). R. F. Bayfield, Anal. Biochem. 39, 282 (1971).
METHODS IN ENZYMOLOGY,VOL. 67
Copyright ~) 1980by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181967-1
190
VITAMIN A GROUP
[24]
0.7
0.6
E 0.5
OJ+
0.3
0.2 .m
0.1
i
1
i
2 Vitamin A
J
3 --
z+ Iag/3m[
5 Fine[
6 Sotufion
7
8
FIG. 1. Relationship between absorbance and vitamin A concentration. From R. E. Bayfield, Anal. Biochem. 39, 282 (]971).
tion. 7 Reference oil (100 mg) is dissolved in chloroform (10 ml). This solution loses about 5% strength after storage for 1 week at - 2 0 °" Vitamin A, weak solution. Dilute the strong solution (1 ml) with chloroform (to 50 ml). This solution contains about 7/~g/ml. Ethanol and light petroleum (b.p. 60/80°), spectroscopic grade.
Procedure PREPARATION OF STANDARD CURVE. To tubes (1 cm in diameter) add aliquots of the weak solution of vitamin A (0.15-1.0 ml) to obtain a 1-7/xg range of vitamin. To each tube add chloroform as required to bring the volume to 1 ml. From a fast-delivery pipette, add trichloroacetic acid reagent (2 ml) rapidly, mixing with the vitamin solution. Read the optical density of the solution at 620 nm in a spectrophotometer or colorimeter at maximum deflection of the galvanometer spot. Plot a graph of optical density at maximum deflection vs vitamin A as /.~g/3 ml final solution, as illustrated in Fig. I. APPLICATION TO SERUM. The serum (15 ml) in a 100-ml stoppered measuring cylinder is shaken with ethanol (15 ml) then with light peTAlternatively, standard preparations of vitamin A alcohol may be used at equivalent concentrations.
[24]
COLORIMETRIC ESTIMATION OF VITAMIN A WITH T C A
191
1.0 •~
p/c~"~~ ~
0.05 ~ TCA
.-~ 0,8
0.1
,o 0.6
~_ 0.4 0
g % O.2
__o__
~o
__--oo.o15
. . . . .
o
a 1.0
O
~ 0.2 o.o15 < O.l+
0.1
c_
0.05
E
O
0.6 o 1.0
~o 0.8 --J
i
,
,
10 Time
,
20
i
i
30
i
i
40
i
J
50
(minufes)
FIG. 2. Effect of concentration of trichloroacetic acid (TCA) on rate of formation of anhydrovitamin A (measured at 377 nm) and loss of vitamin A (measured at 332 nm). Solvent, chloroform. troleum (30 ml) for 1.5-2 min on a mechanical shaker and allowed to stand to obtain a clear supernatant solution. An aliquot (25 ml) of the light petroleum layer is drawn off, the solvent is r e m o v e d under vacuum, and the residue is redissolved in chloroform (1.25 ml). This solution (1 ml) is used for color development with trichloroacetic acid reagent as previously described. APPLICATION TO LIVER TISSUE. The liver (1 g) in a 50-ml beaker with a stout glass rod is finely ground with chromatographic grade silica gel (3-4 g) gradually added, and grinding between additions, until a freeflowing p o w d e r is obtained. The mixture is transferred to a 100-ml stoppered measuring cylinder, shaken mechanically for 30 rain with acetone/ light petroleum, 1 : 1 (50 ml), then allowed to stand 1-1.5 hr to obtain a clear supernatant solution. An aliquot (40 ml) of the solution is drawn off, the solvent is r e m o v e d under vacuum, and the residue is redissolved in
192
VITAMIN A GROUP
[24]
c
0.8
0
0.4
A ~
0.TCA0. 1~'0"2 5
~E o,2 u_
~'0 o
I
,~o..--_--T-,, ,~?--7--,-7 - - - ~
o.ols
~
.015 0.I 0.2
'~ 0.2 U •~ 0.4 .c_ ff
~ 0.6 ~ 0.8 o _._1
10Tim20 e(min30 utes)40
50
FIG. 3. Effect of concentration of trichloroacetic acid (TCA) on rate of formation of anhydrovitamin A (measured at 377 nm) and loss of vitamin A acetate (measured at 332 nm). Solvent, chloroform. chloroform (2 ml). The estimation is then c o m p l e t e d as described for serum, adjusting the aliquot of chloroform solution used for color develo p m e n t as necessary. In practice it m a y be an advisable precaution to carry out duplicate extractions of liver tissue if low vitamin A content is suspected. Discussion The colors produced f r o m vitamin A, its acetate and palmitate with the C a r r - P r i c e antimony trichloride reagent and trichloroacetic acid r e a c h the same intensity for any given a m o u n t of vitamin. H o w e v e r , Fig. 1 shows that color formation is best limited to that f r o m 7/~g/3 ml final solution for accurate s p e c t r o p h o t o m e t r i c m e a s u r e m e n t . The necessity to read at the point of m a x i m u m deflection must be emphasized. A f a s t - m e a s u r e m e n t
[24]
COLORIMETRIC ESTIMATION OF VITAMIN A WITH T C A
193
1.2
1.0
0.8
~U
D
0.6
-6 O
0.4
0.2
250
300
350
/+00
/+50
WaveLength (rim)
FIG. 4. Effect of concentration of trichloroacetic acid (TCA) on the formation and stability of anhydrovitamin A. O O, Vitamin A; [] O, anhydrovitamin from 0.1% TCA; A A, anhydrovitamin from 1% TCA.
accessory and a slave recorder have been described for m e a s u r e m e n t of the m a x i m u m color intensity using the trichloroacetic acid reagent? Tests with dilute trichloroacetic acid reagent allow dissection of the course of reaction and not only clearly establish that the color must be derived f r o m anhydrovitamin A, but also confirm the sensitivity of vitamin A to dehydration even with acid strengths well below that used in the estimation. The concentration of trichloroacetic acid controls the rate of formation and stability of anhydrovitamin, which m a y be correlated with loss of vitamin A or its acetate (Figs. 2 and 3). It is important to o b s e r v e that change occurs without a p p e a r a n c e of blue color in the w e a k e r acid medium. Formation of anhydrovitamin with fine-structure absorption (Fig. 4) follows protonation of vitamin A, and the slower formation f r o m vitamin A acetate reflects differences in the rates of dehydration and deacetoxylation. The catalyzed dehydration of vitamin A has been noted pres. Grys, Analyst 100, 637 (1975).
194
VITAMIN A GROUP
c.3 c.3
c.,
c.,
~
[24]
Fc.3c., CH2OH
c.,
,.
~CH3 H
H
_
L"
"
/
-H
CH3 CH3
CH3
CH3
"~CH 3
J H
CH3 CH3
v
CH3
CH3
CH3 CH3
~
CH3
CH3 CH 2
~CH 3
~CH 3
Products
FIG. 5. Sequence of reactions of vitamin A with acids.
viously9"1° and a method of estimation based on this reaction has been described. H However, it will also be seen (Fig. 4) that there is an area of fine balance between the concentration of trichloroacetic acid catalyzing formation of anhydrovitamin with reasonable stability and that promoting loss in further reactions without appearance of a blue color. The absorbances of anhydrovitamin are derived from the same amount of vitamin A alcohol. Thus, whereas with 0.1% trichloroacetic acid the absorbance of anhydrovitamin at 377 nm is markedly greater than that of vitamin A alcohol at 332 nm, with 1% trichloroacetic acid promotion of further reaction is indicated by the weaker absorbance of the anhydrovitamin. Similar changes occur with vitamin A acetate. Stronger solutions of trichloroacetic acid proceed immediately to protonation of anhydrovitamin, giving the transient blue color of the carbonium ion. Mechanistic aspects of the 9 j. R. Edisbury, A. E. Gillam, I. M. Heilbron, and R. A. Morton, Biochem. J. 26, 1164 (1932). lo E. M. Shantz, G. O. Crawley, and W. D. Embree, J. Am. Chem. Soc. 65, 901 (1965). 11 p. Budowski and A. Bondi, Analyst 82, 571 (1959).
[25]
I N D I R E C T S P E C T R O P H O T O M E T R Y ON V I T A M I N
A
PRODUCTS
195
reaction are indicated in Fig. 5. The spectral properties of this cation and later absorptions in a variety of solvents have been examined, TM and the necessity for high concentrations of antimony trichloride for even brief stabilization of the colored ion in the Carr-Price reaction has been noted. 'a Reaction at lower temperatures markedly affects color stability. If the temperature of both vitamin A solution and trichloroacetic acid reagent is lowered before mixing, the time for measurement is increased from about 5 sec at 20° to 19-20 sec at 10° and 28-30 sec at 0°. Incidental to the temperature reduction loss of strength of the reagent occurs, evidenced by crystallization of trichloroacetic acid, but the fact that there is no loss of color shows that the strength of the reagent is still sufficient to sustain the changes discussed above. Methods of estimation with trifluoroacetic acid as a color-developing reagent using either the acid itself ~ or as a 33% solution in chloroform 14 have been described. The present method employs the less exotic trichloroacetic acid without sacrifice of accuracy. Attention may be drawn to the anomalous properties of glycerol dichlorohydrin as a reagent with alcoholic groups functioning competitively as Lewis bases, thus accelerating decay of color. The requirement for addition of hydrochloric acid to produce "activated" reagent is thus understood .4 With the trichloroacetic acid method, vitamin A concentrations in the range of 1-10/zg/100 ml have been found in sheep serum, restored to 20-30/zg/100 ml after grazing the sheep for 1 month? Liver concentrations as low as 0.2/zg/g have been noted in 2-year-old wethers on a vitamin A-deficient diet and a range 137-453 /zg/g was observed for 1-year-old wethers on green pasture. '5 ,2 p. E. ,3 p. E. ,4 j. B. t5 R. F.
Blatz and D. Pippert, J. Am. Chem. Soc. 90, 1290 (1960). Blatz and D. E s t r a d a , Anal. Chem. 44, 570 (1972). Neeld and W. N. Pearson, J. Nutr. 79, 454 (1963). Bayfield, Anal. Biochem. 64, 403 (1975).
[25] I n d i r e c t S p e c t r o p h o t o m e t r y on V i t a m i n A P r o d u c t s : P e a k Signal R e a d o u t B y S. G R Y S
General Principle The most sensitive reagents known today for determining vitamin A are the Lewis acids antimony(III) chloride, trichloroacetic acid, and
METHODS IN ENZYMOLOGY, VOL. 67
Copyright © 1980by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-1g1967-1
C O L O R I M E T R I C E S T I M A T I O N OF V I T A M I N A W I T H T C A
[2 4] C o l o r i m e t r i c
189
Estimation of Vitamin A with Trichloroacetic Acid
By R. F. BAYFIELD and E. R. COLE It is now over 50 years since the color reaction of vitamin A with strong acids, notably sulfuric acid, was first observed. 1'2 The importance of this test lies in the fact that it provided the basis for a general view of the reaction of vitamin A with Lewis acids, including the Carr-Price antimony trichloride reaction,3 which has tended to dominate colorimetric methods of estimation from that time. However, several unsatisfactory features of the Carr-Price method, particularly interference by moisture and instability of the color, have prompted continued investigations for alternative reagents. Of these, glycerol dichlorohydrin, 4 trifluoroacetic acidp and trichloroacetic acid 6 have received most prominence, but development of methods has proceeded in an empirical manner and only comparatively recently have mechanistic aspects of these reactions received consideration. It is in this context that the trichloroacetic acid method is now discussed with particular reference to estimation of vitamin A in serum and liver tissue. Experimental
Reagents. All reagents should be freshly prepared. Exposure of samples and reagents to light must be avoided at all times. Trichloroacetic acid, saturated solution in chloroform. Trichloroacetic acid crystals (50 g), freshly washed with solvent, are dissolved in alcohol-flee distilled chloroform (25 ml). This gives about 50 ml of reagent which is stable for 5 hr with daylight excluded. Optical density readings tend to decrease with storage for longer periods, up to 24 hr. No color is given by vitamin A with reagent exposed to bright diffuse sunlight for several hours. Vitamin A (U.S.P. Reference capsule vitamin A acetate), strong solu-
1 j. C. D r u m m o n d and A. F. Watson, Analyst 47, 341 (1922). 2 0 . Rosenheim and J. C. Drummond, Biochem. J. 19, 753 (1925). '~ F. H. Carr and E. A. Price, Biochem. J. 20, 497 (1926). 4 A. E. Sobel and H. Werbin, J. Biol. Chem. 159, 681 (1945);Ind. Eng. Chem. Anal. Ed. 18, 570 (1946). 5 R. E. Dugan, N. A. Frigerio, and J. M. Siebert, Anal. Chem. 36, 114 (1964). R. F. Bayfield, Anal. Biochem. 39, 282 (1971).
METHODS IN ENZYMOLOGY,VOL. 67
Copyright ~) 1980by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181967-1
190
VITAMIN A GROUP
[24]
0.7
0.6
E 0.5
OJ+
0.3
0.2 .m
0.1
i
1
i
2 Vitamin A
J
3 --
z+ Iag/3m[
5 Fine[
6 Sotufion
7
8
FIG. 1. Relationship between absorbance and vitamin A concentration. From R. E. Bayfield, Anal. Biochem. 39, 282 (]971).
tion. 7 Reference oil (100 mg) is dissolved in chloroform (10 ml). This solution loses about 5% strength after storage for 1 week at - 2 0 °" Vitamin A, weak solution. Dilute the strong solution (1 ml) with chloroform (to 50 ml). This solution contains about 7/~g/ml. Ethanol and light petroleum (b.p. 60/80°), spectroscopic grade.
Procedure PREPARATION OF STANDARD CURVE. To tubes (1 cm in diameter) add aliquots of the weak solution of vitamin A (0.15-1.0 ml) to obtain a 1-7/xg range of vitamin. To each tube add chloroform as required to bring the volume to 1 ml. From a fast-delivery pipette, add trichloroacetic acid reagent (2 ml) rapidly, mixing with the vitamin solution. Read the optical density of the solution at 620 nm in a spectrophotometer or colorimeter at maximum deflection of the galvanometer spot. Plot a graph of optical density at maximum deflection vs vitamin A as /.~g/3 ml final solution, as illustrated in Fig. I. APPLICATION TO SERUM. The serum (15 ml) in a 100-ml stoppered measuring cylinder is shaken with ethanol (15 ml) then with light peTAlternatively, standard preparations of vitamin A alcohol may be used at equivalent concentrations.
[24]
COLORIMETRIC ESTIMATION OF VITAMIN A WITH T C A
191
1.0 •~
p/c~"~~ ~
0.05 ~ TCA
.-~ 0,8
0.1
,o 0.6
~_ 0.4 0
g % O.2
__o__
~o
__--oo.o15
. . . . .
o
a 1.0
O
~ 0.2 o.o15 < O.l+
0.1
c_
0.05
E
O
0.6 o 1.0
~o 0.8 --J
i
,
,
10 Time
,
20
i
i
30
i
i
40
i
J
50
(minufes)
FIG. 2. Effect of concentration of trichloroacetic acid (TCA) on rate of formation of anhydrovitamin A (measured at 377 nm) and loss of vitamin A (measured at 332 nm). Solvent, chloroform. troleum (30 ml) for 1.5-2 min on a mechanical shaker and allowed to stand to obtain a clear supernatant solution. An aliquot (25 ml) of the light petroleum layer is drawn off, the solvent is r e m o v e d under vacuum, and the residue is redissolved in chloroform (1.25 ml). This solution (1 ml) is used for color development with trichloroacetic acid reagent as previously described. APPLICATION TO LIVER TISSUE. The liver (1 g) in a 50-ml beaker with a stout glass rod is finely ground with chromatographic grade silica gel (3-4 g) gradually added, and grinding between additions, until a freeflowing p o w d e r is obtained. The mixture is transferred to a 100-ml stoppered measuring cylinder, shaken mechanically for 30 rain with acetone/ light petroleum, 1 : 1 (50 ml), then allowed to stand 1-1.5 hr to obtain a clear supernatant solution. An aliquot (40 ml) of the solution is drawn off, the solvent is r e m o v e d under vacuum, and the residue is redissolved in
192
VITAMIN A GROUP
[24]
c
0.8
0
0.4
A ~
0.TCA0. 1~'0"2 5
~E o,2 u_
~'0 o
I
,~o..--_--T-,, ,~?--7--,-7 - - - ~
o.ols
~
.015 0.I 0.2
'~ 0.2 U •~ 0.4 .c_ ff
~ 0.6 ~ 0.8 o _._1
10Tim20 e(min30 utes)40
50
FIG. 3. Effect of concentration of trichloroacetic acid (TCA) on rate of formation of anhydrovitamin A (measured at 377 nm) and loss of vitamin A acetate (measured at 332 nm). Solvent, chloroform. chloroform (2 ml). The estimation is then c o m p l e t e d as described for serum, adjusting the aliquot of chloroform solution used for color develo p m e n t as necessary. In practice it m a y be an advisable precaution to carry out duplicate extractions of liver tissue if low vitamin A content is suspected. Discussion The colors produced f r o m vitamin A, its acetate and palmitate with the C a r r - P r i c e antimony trichloride reagent and trichloroacetic acid r e a c h the same intensity for any given a m o u n t of vitamin. H o w e v e r , Fig. 1 shows that color formation is best limited to that f r o m 7/~g/3 ml final solution for accurate s p e c t r o p h o t o m e t r i c m e a s u r e m e n t . The necessity to read at the point of m a x i m u m deflection must be emphasized. A f a s t - m e a s u r e m e n t
[24]
COLORIMETRIC ESTIMATION OF VITAMIN A WITH T C A
193
1.2
1.0
0.8
~U
D
0.6
-6 O
0.4
0.2
250
300
350
/+00
/+50
WaveLength (rim)
FIG. 4. Effect of concentration of trichloroacetic acid (TCA) on the formation and stability of anhydrovitamin A. O O, Vitamin A; [] O, anhydrovitamin from 0.1% TCA; A A, anhydrovitamin from 1% TCA.
accessory and a slave recorder have been described for m e a s u r e m e n t of the m a x i m u m color intensity using the trichloroacetic acid reagent? Tests with dilute trichloroacetic acid reagent allow dissection of the course of reaction and not only clearly establish that the color must be derived f r o m anhydrovitamin A, but also confirm the sensitivity of vitamin A to dehydration even with acid strengths well below that used in the estimation. The concentration of trichloroacetic acid controls the rate of formation and stability of anhydrovitamin, which m a y be correlated with loss of vitamin A or its acetate (Figs. 2 and 3). It is important to o b s e r v e that change occurs without a p p e a r a n c e of blue color in the w e a k e r acid medium. Formation of anhydrovitamin with fine-structure absorption (Fig. 4) follows protonation of vitamin A, and the slower formation f r o m vitamin A acetate reflects differences in the rates of dehydration and deacetoxylation. The catalyzed dehydration of vitamin A has been noted pres. Grys, Analyst 100, 637 (1975).
194
VITAMIN A GROUP
c.3 c.3
c.,
c.,
~
[24]
Fc.3c., CH2OH
c.,
,.
~CH3 H
H
_
L"
"
/
-H
CH3 CH3
CH3
CH3
"~CH 3
J H
CH3 CH3
v
CH3
CH3
CH3 CH3
~
CH3
CH3 CH 2
~CH 3
~CH 3
Products
FIG. 5. Sequence of reactions of vitamin A with acids.
viously9"1° and a method of estimation based on this reaction has been described. H However, it will also be seen (Fig. 4) that there is an area of fine balance between the concentration of trichloroacetic acid catalyzing formation of anhydrovitamin with reasonable stability and that promoting loss in further reactions without appearance of a blue color. The absorbances of anhydrovitamin are derived from the same amount of vitamin A alcohol. Thus, whereas with 0.1% trichloroacetic acid the absorbance of anhydrovitamin at 377 nm is markedly greater than that of vitamin A alcohol at 332 nm, with 1% trichloroacetic acid promotion of further reaction is indicated by the weaker absorbance of the anhydrovitamin. Similar changes occur with vitamin A acetate. Stronger solutions of trichloroacetic acid proceed immediately to protonation of anhydrovitamin, giving the transient blue color of the carbonium ion. Mechanistic aspects of the 9 j. R. Edisbury, A. E. Gillam, I. M. Heilbron, and R. A. Morton, Biochem. J. 26, 1164 (1932). lo E. M. Shantz, G. O. Crawley, and W. D. Embree, J. Am. Chem. Soc. 65, 901 (1965). 11 p. Budowski and A. Bondi, Analyst 82, 571 (1959).
[25]
I N D I R E C T S P E C T R O P H O T O M E T R Y ON V I T A M I N
A
PRODUCTS
195
reaction are indicated in Fig. 5. The spectral properties of this cation and later absorptions in a variety of solvents have been examined, TM and the necessity for high concentrations of antimony trichloride for even brief stabilization of the colored ion in the Carr-Price reaction has been noted. 'a Reaction at lower temperatures markedly affects color stability. If the temperature of both vitamin A solution and trichloroacetic acid reagent is lowered before mixing, the time for measurement is increased from about 5 sec at 20° to 19-20 sec at 10° and 28-30 sec at 0°. Incidental to the temperature reduction loss of strength of the reagent occurs, evidenced by crystallization of trichloroacetic acid, but the fact that there is no loss of color shows that the strength of the reagent is still sufficient to sustain the changes discussed above. Methods of estimation with trifluoroacetic acid as a color-developing reagent using either the acid itself ~ or as a 33% solution in chloroform 14 have been described. The present method employs the less exotic trichloroacetic acid without sacrifice of accuracy. Attention may be drawn to the anomalous properties of glycerol dichlorohydrin as a reagent with alcoholic groups functioning competitively as Lewis bases, thus accelerating decay of color. The requirement for addition of hydrochloric acid to produce "activated" reagent is thus understood .4 With the trichloroacetic acid method, vitamin A concentrations in the range of 1-10/zg/100 ml have been found in sheep serum, restored to 20-30/zg/100 ml after grazing the sheep for 1 month? Liver concentrations as low as 0.2/zg/g have been noted in 2-year-old wethers on a vitamin A-deficient diet and a range 137-453 /zg/g was observed for 1-year-old wethers on green pasture. '5 ,2 p. E. ,3 p. E. ,4 j. B. t5 R. F.
Blatz and D. Pippert, J. Am. Chem. Soc. 90, 1290 (1960). Blatz and D. E s t r a d a , Anal. Chem. 44, 570 (1972). Neeld and W. N. Pearson, J. Nutr. 79, 454 (1963). Bayfield, Anal. Biochem. 64, 403 (1975).
[25] I n d i r e c t S p e c t r o p h o t o m e t r y on V i t a m i n A P r o d u c t s : P e a k Signal R e a d o u t B y S. G R Y S
General Principle The most sensitive reagents known today for determining vitamin A are the Lewis acids antimony(III) chloride, trichloroacetic acid, and
METHODS IN ENZYMOLOGY, VOL. 67
Copyright © 1980by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-1g1967-1