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A note on a new concentration of antimony trichloride reagent in the U. S. P. XIV identification test for vitamin A
Notes
A Note on a New Concentration of Antimony Trichloride Reagent in the U. S. P. XIV Identification Test for Vitamin A* By JAMES A. BROWN
T
HE vitamin A identification test (Carr-Price test) which forms a part of the U. S. P. XIV assay for vitamin A (1) has been the subject of much study by many workers. The fleeting nature of the blue color formed is well known and constitutes a major obstacle t o the utilization of this identification procedure. To our knowledge, no work has been reported on studies of the reaction of vitamin A with concentrations of antimony trichloride reagent lower than specified. Work in this laboratory has shown that the speed of the development t o peak intensity of the vitamin A-SbCLp blue color can be decreased t o a point where there is ample time to determine a readable maximum by use of a 6 Gm./lOO cc. reagent instead of the 20 Gm./lOO cc. Repeated determinations using both reagents have shown that the calibration curves obtained with the U. S. P. reference standard are similar in shape and degree of linearity, the difference being that as the reagent concentration decreases, the slope of the useful range decreases. Further, as long as the same reagent concentration is used for both standard and unknown, the results obtained for unknowns with either strength are identical except for variations in the results due t o the inability t o reproduce the readings as well with the stronger reagent as with the weaker. Figure 1 shows the family of calibration curves resulting when various strengths of reagent are used, and Table I indicates the length of time required for the blue color to reach a maximum in-
TABLE I Strength of Reagent, Gm./100 Cc.
Time to Reach Peak Intensity, Sec.
20 10 6
1 3
3
13
7
tensity when different strengths of reagent are used. Consideration of both the slope of the curve and the time data leads to the conclusion that the optimum reagent strength is about 6 Gm./100 cc. Strengths much below this give absorbancies below the 0.2 to 0.5 useful range specified by the U. S. P. XIV.
0.800 -
K
0
-1
8
0.600-
W 2 1
m LL
0
5
0.400
-
m a E
s!m a
0.200
0
0
20 40 60 80 UNITS OF VITAMIN A
Fig. 1.-Calibration
100
curves with various reagent strengths.
When the weaker reagents are used, the characteristic red color which follows the blue color appears earlier. However, even with the weakest reagent, the red appears after the blue color maximum, and, so far as we have been able t o determine, it has no absorption effect at 620 mp. Both a modified Coleman Spectrophotometer and a Model B Beckman Spectrophotometer have been used in this laboratory for these measurements. Other advantages of the weaker reagent are that it is less corrosive, making i t easier t o handle and dispense, and that it is less expensive t o use. It is hoped that other laboratories will gather data of their own using the weaker reagent, and that its general usefulness will become established t o the end that it may become part of the official method. REFERENCE
* Received
September 25, 1950, from the Analytical Laboratories, Eli Lilly and Co., Indianapolis, Ind.
(1) “United States Pharmacopeia,” Fourteenth Revision. Mack Publishing Co., Easton, Pa., 1950.
699
A Note on a New Concentration of Antimony Trichloride Reagent in the U. S. P. XIV Identification Test for Vitamin A* By JAMES A. BROWN
T
HE vitamin A identification test (Carr-Price test) which forms a part of the U. S. P. XIV assay for vitamin A (1) has been the subject of much study by many workers. The fleeting nature of the blue color formed is well known and constitutes a major obstacle t o the utilization of this identification procedure. To our knowledge, no work has been reported on studies of the reaction of vitamin A with concentrations of antimony trichloride reagent lower than specified. Work in this laboratory has shown that the speed of the development t o peak intensity of the vitamin A-SbCLp blue color can be decreased t o a point where there is ample time to determine a readable maximum by use of a 6 Gm./lOO cc. reagent instead of the 20 Gm./lOO cc. Repeated determinations using both reagents have shown that the calibration curves obtained with the U. S. P. reference standard are similar in shape and degree of linearity, the difference being that as the reagent concentration decreases, the slope of the useful range decreases. Further, as long as the same reagent concentration is used for both standard and unknown, the results obtained for unknowns with either strength are identical except for variations in the results due t o the inability t o reproduce the readings as well with the stronger reagent as with the weaker. Figure 1 shows the family of calibration curves resulting when various strengths of reagent are used, and Table I indicates the length of time required for the blue color to reach a maximum in-
TABLE I Strength of Reagent, Gm./100 Cc.
Time to Reach Peak Intensity, Sec.
20 10 6
1 3
3
13
7
tensity when different strengths of reagent are used. Consideration of both the slope of the curve and the time data leads to the conclusion that the optimum reagent strength is about 6 Gm./100 cc. Strengths much below this give absorbancies below the 0.2 to 0.5 useful range specified by the U. S. P. XIV.
0.800 -
K
0
-1
8
0.600-
W 2 1
m LL
0
5
0.400
-
m a E
s!m a
0.200
0
0
20 40 60 80 UNITS OF VITAMIN A
Fig. 1.-Calibration
100
curves with various reagent strengths.
When the weaker reagents are used, the characteristic red color which follows the blue color appears earlier. However, even with the weakest reagent, the red appears after the blue color maximum, and, so far as we have been able t o determine, it has no absorption effect at 620 mp. Both a modified Coleman Spectrophotometer and a Model B Beckman Spectrophotometer have been used in this laboratory for these measurements. Other advantages of the weaker reagent are that it is less corrosive, making i t easier t o handle and dispense, and that it is less expensive t o use. It is hoped that other laboratories will gather data of their own using the weaker reagent, and that its general usefulness will become established t o the end that it may become part of the official method. REFERENCE
* Received
September 25, 1950, from the Analytical Laboratories, Eli Lilly and Co., Indianapolis, Ind.
(1) “United States Pharmacopeia,” Fourteenth Revision. Mack Publishing Co., Easton, Pa., 1950.
699