ANALYTICAL
BIOCHEMISTRY
96,
l-5
(1979)
Spectrophotometric HUI-HUA Department
of Chemistry,
The Ohio
Determination SHIEH State
AND THOMAS
University.
140 West
of Ascorbic
Acid
R. SWEET 18th Avenue,
Columbus,
Ohio
43210
Received June 29, 1978 A spectrophotometric method for determining ascorbic acid based on the redox reaction between a copper (II)-2,2’-biquinoline solution and ascorbic acid was developed. The purple color of the copper (I)-2,2’-biquinoline complex formed in a buffered acetone-water solution was measured at 540 nm. Minerals, sugars, and other vitamins do not interfere when present in quantities usually found in pharmaceutical preparations. Ferrous interference was eliminated by treating the sample solution with the cation-exchange resin Dowex 5OWX12. Several single component and multivitamin formulations were satisfactorily analyzed by this method. Its high sensitivity permits measurements of quantities of ascorbic acid to 3.4 pg/ml of sample solution. The procedure is simple, rapid, and suitable for routine control.
2,2’-Biquinoline (biq)’ is widely used as a reagent for the spectrophotometric determination of copper (I) because of its specificity and sensitivity (l-4). The determinations are based on the formation of the purple bis-biq-copper (I) complex which is extracted into an immiscible organic solvent such as n-hexyl alcohol. In determinations of copper (II), a reducing agent is needed to reduce copper (II) to copper (I). Hydroxylamine hydrochloride is usually used for this purpose. In a study of copper (II) and copper (I)-2,2’-biq complexes, Faye found that the addition of an excess of ascorbic acid to a water-acetone solution containing copper (II) and biq brings about essentially complete formation of the bis-biq-copper (I) complex (5). The rapid reaction of ascorbic acid with an excess of a copper (II)-biq solution is the basis of the proposed method of analysis of ascorbic acid.
EXPERIMENTAL Reagents
All reagents used were analytical grade. Aqueous solutions were prepared using demineralized double-distilled water provided by the Ohio State University Reagent Laboratory. Oxygen-free solutions were prepared by purging with nitrogen. Phosphate -citric acid buffer. The pH of a 0.01 M citric acid solution was adjusted to pH 3.00 by adding solid disodium hydrogen phosphate. Reagent solution. One hundred milliliters of CtP+ solution (0.13 g CuC1,*2H,0/500 ml buffer) was mixed with 300 ml of biq solution (0.37 g biq/lOOO ml acetone). Standard ascorbic acid solution. A standard solution was prepared by accurately weighing 0.05 g of ascorbic acid, dissolving it in deaerated buffer, and diluting to 100 ml with deaerated buffer. Sample solutions. Sample solutions were prepared from commercial vitamin tablets or capsules by stirring the tablets or the
1 Abbreviations used: biq, 2,2’-Biquinoline; AsA, ascorbic acid; CBQ, copper biquinoline; DCPIP, 2,6dichlorophenol indophenol.
1
0003-2697/79/090001-05$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
2
SHIEH AND SWEET
powder from the capsules in deaerated buffer until solution was complete and diluting to a known volume with the deaerated buffer. The final solutions contained 100 to 300 pg!rnl of ascorbic acid.
0.40
-
cE g 0.30-
Procedure
In each 25ml volumetric flask, 20 ml of Cu(biq),2+ reagent solution and a different known volume of the standard ascorbic acid solution were added. The solution was diluted to the mark with buffer. A blank solution was prepared by diluting 20 ml of the reagent solution to 25 ml with buffer. Absorbances were measured at 540 nm and a calibration curve was plotted. For the unknown determinations, 1 ml of the sample solution was added in place of the standard ascorbic acid solution. For the analysis of unknown samples containing up to 40 mg of iron as ferrous salt, 2 g of Dowex 5OW-X 12 in the hydrogen form was added to the sample solution. The mixture was stirred by bubbling nitrogen through it for 2 min. After the resin beads settled, the supernatant liquid was analyzed immediately according to the procedure given above. RESULTS AND DISCUSSION Although solid ascorbic acid is stable in air when dry, ascorbic acid solutions react easily with molecular oxygen from the air. Furthermore, trace amounts of metal ions such as Cu2+ or Fe3+ catalyze the oxidation (6). Both the uncatalyzed and catalyzed oxidations are pH dependent. Therefore, in order to preserve the ascorbic acid in solution, it is necessary to deoxygenate the demineralized double-distilled water with Nz before the addition of the ascorbic acid. Figure 1 shows the effect of pH on a solution of ascorbic acid in deaerated phosphatecitric acid buffer that was analyzed immediately after preparation and also on a similarly prepared solution that was allowed to
0
I 3
I 4
I 5
I 6
PH
FIG. 1. pH effect on the stability of the ascorbic acid solution. Solution of AsA in deaerated buffer {O) immediately after preparation and (A) after exposure to air for I h.
stand exposed to air for 1 hour before analysis. In the proposed method, the purplecolored Cu(biq),+ in the final solution is quite stable. The absorbance measured at 540 nm does not change for at least 1 h after mixing the unknown solution with the reagent and buffer. Using the proposed procedure, the calibration curve (A%,, plotted against ascorbic acid concentration in the sample solution) is a straight line up to 27.2 mg ascorbic acid/ 100 ml sample solution, where the absorbance is 0.80. The sensitivity, defined as the concentration of ascorbic acid required to give an absorbance of 0.05, is 17 p&ml. However, if the suggested procedure is modified by replacing the 25-ml volumetric flask with a 5-ml flask and the aliquot of ascorbic acid sample sofution added to the flask is unchanged, the sensitivity is 3.4 dml. The widely used 2,6-dichlorophenol indophenol method for ascorbic acid analysis is based on the conversion of a colored reagent to a colorless compound on reaction with ascorbic acid (7-9). It has two disadvantages compared with the proposed method. First, very careful timing is necessary in the 2,6-dichlorophenol indophenol
SPECTROPHOTOMETRIC
DETERMINATION
method because of the fading of the colored reagent under the final reaction conditions. Second, since the decrease in absorption of the colored reagent is measured, considerably more care must be exercised in the addition of reagent solution to both unknown and blank solutions. A series of pure ascorbic acid solutions with various concentrations were analyzed by the proposed copper biquinolone (CBQ) method and the 2,6-dichlorophenol indophenol (DCPIP) method. Table 1 shows that the results by both methods agree well. The effect of the presence of other vitamins, minerals, sugars, and common reducing agents that are likely to be present along with ascorbic acid in foodstuffs or
OF ASCORBIC TABLE
ACID 1
DETERMINATION OF AsA IN ASCORBIC ACID SOLUTIONS
PURE
AsA found (mg1100 ml) AsA added (mg1100 ml) 12.1 17.6 19.2 22.0
CBQ method 11.95 17.60 19.35 22.05
DCPIP method
-c 0.05 + 0.00 k 0.15 2 0.05
a Average of two determinations, ation from the mean.
12.10 17.50 19.15 22.25
pharmaceutical products is shown in Table 2. Most of the substances tested for interference, even when present in a concentra-
OF ASCORBIC ACID BY THE CBQ METHOD IN THE PRESENCE OF MINERALS, VITAMINS, REDUCING SUGARS, AND COMMON REDUCING AGENTS
Substance
MgSO, ZnSO,.7H,O Co(N0&.6H20 K,SO, MnSO, 3H,O Thiamine. HCl Nicotinamide Riboflavin Nicotinic acid Pantothenic acid Pyridoxine Fructose Glucose Lactose D-Mannose Sucrose Hydroxylamine-HCl Hydrazine-H,SO, Urea Thiourea Cysteine Glutathione Sodium thiosulfate FeSOI.7H,0 a Average of two determinations,
0.10 0.05 0.15 0.05
with average devi-
TABLE DETERMINATION OTHER
-t k -+ -t
Amount added (mg)
AsA added (ms)
1000 1000 1000 1000 1000 850 1000 10 1000 1000 1000 1000 1000 1000 1000 1000 200 100 1000 10 10 10 10 10
10.1 10.1 10.1 10.1 10.1 10.2 10.2 10.2 10.2 10.2 10.2 10.1 10.1 10.1 10.1 10.1 10.1 10.1 10.1 10.1 10.1 10.1 10.1 10.1
with average deviation from the mean.
AsA found” (mg) 10.15 10.15 10.00 10.20 10.10 10.25 10.20 10.15 10.10 10.20 10.20 10.05 10.05 10.10 10.20 10.10 10.30 10.05 10.00 10.05 15.20 12.70 13.05 13.05
t 0.05 f 0.05 2 0.00 k 0.10 t 0.10 2 0.05 f 0.10 k 0.05 + 0.00 -r- 0.10 + 0.10 2 0.05 + 0.05 * 0.10 r 0.00 f 0.10 k 0.05 +- 0.05 2 0.00 r 0.05 c 0.05 f 0.05 k 0.05 + 0.05
4
SHIEH AND SWEET TABLE
3
DETERMINATIONOFASCORBICACIDINTHEPRESENCE OF Fe*+ WITH AND WITHOUTDOWEX 5OW-X12 TREATMENT
FeSO,.7H,O added (md 40.0 40.0 40.0 40.0 0.0
AsA added (mg)
Dowex SOW-Xl2 added (Id
0.0 0.0 0.0 9.5 9.2
0.00 1.00 2.00 2.00 2.00
AsA found (mg)” 12.8 3.3 0.0 9.4 9.0
a Average of two determinations, ation from the mean.
+- 0.1 -c 0.0 c 0.0 2 0.2 5 0.1
with average devi-
tion as high as lOO-fold that of ascorbic acid, do not cause any interference. Either riboflavin or thiourea will give higher absorbance values ifpresent in more than an equal amount of ascorbic acid, but the quantities normally encountered for both of these substances are usually lower than this limit. Hydroxylamine-HCl may be present in quantities up to 20-fold that of ascorbic acid and hydrazine-H&SO, may be present in quantities up to IO-fold that of ascorbic acid without interference. The substances found to interfere in the analysis of ascorbic acid by the proposed method include sodium thiosulfate, ferrous sulfate, and sulfhydryl compounds such as TABLE
4
DETERMINATION OF ASCORBIC PHARMACEUTICALPRODUCTS
Tablet No.
AsA claimed (mg)
I II III IV V
250 250 300 60 60
ACID
IN
AsA found (mg)
CBQ 247.4 248.0 316.4 59.2 53.2
r + k + 2
n Average of three determinations ation from the mean.
DCPIP 0.6 0.8 0.4 0.2 0.2
244.0 246.6 318.4 60.5 54.2
f 1.4 ZII 0.8 + 0.8 2 0.5 + 0.6
with average devi-
cysteine and glutathione. These substances also interfere with the 2,6-dichlorophenol indophenol method (10). Van Eekelen and Emmerie removed sulfhydryl compounds and thiosulfate with mercuric acetate prior to analysis for ascorbic acid with 2,6dichlorophenol indophenol(l1). These substances are precipitated by mercuric acetate whereas the ascorbic acid remains in solution as dehydroascorbic acid. To remove the excess mercuric acetate after the precipitation and to reduce the dehydroascorbit acid back to ascorbic acid, H,S was used. Formaldehyde condensation was recommended by Lugg and Mapson to differentiate ascorbic acid from sulfite, thiosulfate, sufhydryl compounds, reductones, and reductic acid (12,13). In the present work it was found that the interference of ferrous sulfate, which is frequently present in pharmaceutical products, can be prevented by the use of the cationexchange resin Dowex 5OW-X12. For the analysis of Fez+-containing samples, 2 g of Dowex 5OW-X12 in the H+ form is sufficient to remove the iron in as much as 40 mg of FeSO, .7H,O present in ascorbic acid samples. Results are shown in Table 3. The proposed method has been applied to the determination of ascorbic acid in pharmaceutical products. Table 4 shows the results obtained for the analysis of the ascorbic acid content in five commercial vitamin products. Tablets I and II are vitamin C tablets. Sample III is a multivitamin capsule. Tablets IV and V are multivitamin preparations that also contain ferrous salt. Each sample solution of tablets IV and V was treated with the cation-exchange resin prior to analysis. REFERENCES 1. Hoste, J., Eeckhout, J., and Gillis, J. (1953)Anal. Chim.
Acta
9, 263-274.
2. Guest, J. (1953) Anal. Chem. 25, 1484- 1486. 3. Ptlaum, R. T., Popov, A. L., and Goodspeed, N. C. (1955) Anal. Chem. 27, 253-255.
SPECTROPHOTOMETRIC
DETERMINATION
4. Bahr, H., and Lipinska, I. (1968) Gem. Anal. (Warsaw) 13(2), 399-403. 5. Faye, G. H. (1966) Canad. J. Chem. 44, 27292736. 6. Taqui Khan, M. M., and Martell, A. E. (1967) J. Amer. Chem. Sot. 89, 4176-4185. 7. Bessey, 0. A. (1938)J. Biol. Chem. 126,771-784. 8. Morell, S. A. (1941) Anal. Chem. 13, 793-794. 9. LoeffIer, H. J., and Ponting, J. D. (1942) Anal. Chem. 14, 846-849.
OF ASCORBIC
5
ACID
IO. Sebrell, W. H., and Harris, R. S. (1967) The Vitamins, 2nd ed., Vol. 1, pp. 345-352, Academic Press, New York. 11. Van Eekelen, M., and Emmerie, A. (1936) Biothem.
J. 30, 25-27.
12. Lug, J. W. H. (1942) 20, 273-285. 13. Mapson, L. W. (1943)J. 62, 223-232.
Ausr.
Sot.
J. Exp.
Chem.
Ind.
Biol.
Med.
(London)
BIOCHEMISTRY
96,
l-5
(1979)
Spectrophotometric HUI-HUA Department
of Chemistry,
The Ohio
Determination SHIEH State
AND THOMAS
University.
140 West
of Ascorbic
Acid
R. SWEET 18th Avenue,
Columbus,
Ohio
43210
Received June 29, 1978 A spectrophotometric method for determining ascorbic acid based on the redox reaction between a copper (II)-2,2’-biquinoline solution and ascorbic acid was developed. The purple color of the copper (I)-2,2’-biquinoline complex formed in a buffered acetone-water solution was measured at 540 nm. Minerals, sugars, and other vitamins do not interfere when present in quantities usually found in pharmaceutical preparations. Ferrous interference was eliminated by treating the sample solution with the cation-exchange resin Dowex 5OWX12. Several single component and multivitamin formulations were satisfactorily analyzed by this method. Its high sensitivity permits measurements of quantities of ascorbic acid to 3.4 pg/ml of sample solution. The procedure is simple, rapid, and suitable for routine control.
2,2’-Biquinoline (biq)’ is widely used as a reagent for the spectrophotometric determination of copper (I) because of its specificity and sensitivity (l-4). The determinations are based on the formation of the purple bis-biq-copper (I) complex which is extracted into an immiscible organic solvent such as n-hexyl alcohol. In determinations of copper (II), a reducing agent is needed to reduce copper (II) to copper (I). Hydroxylamine hydrochloride is usually used for this purpose. In a study of copper (II) and copper (I)-2,2’-biq complexes, Faye found that the addition of an excess of ascorbic acid to a water-acetone solution containing copper (II) and biq brings about essentially complete formation of the bis-biq-copper (I) complex (5). The rapid reaction of ascorbic acid with an excess of a copper (II)-biq solution is the basis of the proposed method of analysis of ascorbic acid.
EXPERIMENTAL Reagents
All reagents used were analytical grade. Aqueous solutions were prepared using demineralized double-distilled water provided by the Ohio State University Reagent Laboratory. Oxygen-free solutions were prepared by purging with nitrogen. Phosphate -citric acid buffer. The pH of a 0.01 M citric acid solution was adjusted to pH 3.00 by adding solid disodium hydrogen phosphate. Reagent solution. One hundred milliliters of CtP+ solution (0.13 g CuC1,*2H,0/500 ml buffer) was mixed with 300 ml of biq solution (0.37 g biq/lOOO ml acetone). Standard ascorbic acid solution. A standard solution was prepared by accurately weighing 0.05 g of ascorbic acid, dissolving it in deaerated buffer, and diluting to 100 ml with deaerated buffer. Sample solutions. Sample solutions were prepared from commercial vitamin tablets or capsules by stirring the tablets or the
1 Abbreviations used: biq, 2,2’-Biquinoline; AsA, ascorbic acid; CBQ, copper biquinoline; DCPIP, 2,6dichlorophenol indophenol.
1
0003-2697/79/090001-05$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
2
SHIEH AND SWEET
powder from the capsules in deaerated buffer until solution was complete and diluting to a known volume with the deaerated buffer. The final solutions contained 100 to 300 pg!rnl of ascorbic acid.
0.40
-
cE g 0.30-
Procedure
In each 25ml volumetric flask, 20 ml of Cu(biq),2+ reagent solution and a different known volume of the standard ascorbic acid solution were added. The solution was diluted to the mark with buffer. A blank solution was prepared by diluting 20 ml of the reagent solution to 25 ml with buffer. Absorbances were measured at 540 nm and a calibration curve was plotted. For the unknown determinations, 1 ml of the sample solution was added in place of the standard ascorbic acid solution. For the analysis of unknown samples containing up to 40 mg of iron as ferrous salt, 2 g of Dowex 5OW-X 12 in the hydrogen form was added to the sample solution. The mixture was stirred by bubbling nitrogen through it for 2 min. After the resin beads settled, the supernatant liquid was analyzed immediately according to the procedure given above. RESULTS AND DISCUSSION Although solid ascorbic acid is stable in air when dry, ascorbic acid solutions react easily with molecular oxygen from the air. Furthermore, trace amounts of metal ions such as Cu2+ or Fe3+ catalyze the oxidation (6). Both the uncatalyzed and catalyzed oxidations are pH dependent. Therefore, in order to preserve the ascorbic acid in solution, it is necessary to deoxygenate the demineralized double-distilled water with Nz before the addition of the ascorbic acid. Figure 1 shows the effect of pH on a solution of ascorbic acid in deaerated phosphatecitric acid buffer that was analyzed immediately after preparation and also on a similarly prepared solution that was allowed to
0
I 3
I 4
I 5
I 6
PH
FIG. 1. pH effect on the stability of the ascorbic acid solution. Solution of AsA in deaerated buffer {O) immediately after preparation and (A) after exposure to air for I h.
stand exposed to air for 1 hour before analysis. In the proposed method, the purplecolored Cu(biq),+ in the final solution is quite stable. The absorbance measured at 540 nm does not change for at least 1 h after mixing the unknown solution with the reagent and buffer. Using the proposed procedure, the calibration curve (A%,, plotted against ascorbic acid concentration in the sample solution) is a straight line up to 27.2 mg ascorbic acid/ 100 ml sample solution, where the absorbance is 0.80. The sensitivity, defined as the concentration of ascorbic acid required to give an absorbance of 0.05, is 17 p&ml. However, if the suggested procedure is modified by replacing the 25-ml volumetric flask with a 5-ml flask and the aliquot of ascorbic acid sample sofution added to the flask is unchanged, the sensitivity is 3.4 dml. The widely used 2,6-dichlorophenol indophenol method for ascorbic acid analysis is based on the conversion of a colored reagent to a colorless compound on reaction with ascorbic acid (7-9). It has two disadvantages compared with the proposed method. First, very careful timing is necessary in the 2,6-dichlorophenol indophenol
SPECTROPHOTOMETRIC
DETERMINATION
method because of the fading of the colored reagent under the final reaction conditions. Second, since the decrease in absorption of the colored reagent is measured, considerably more care must be exercised in the addition of reagent solution to both unknown and blank solutions. A series of pure ascorbic acid solutions with various concentrations were analyzed by the proposed copper biquinolone (CBQ) method and the 2,6-dichlorophenol indophenol (DCPIP) method. Table 1 shows that the results by both methods agree well. The effect of the presence of other vitamins, minerals, sugars, and common reducing agents that are likely to be present along with ascorbic acid in foodstuffs or
OF ASCORBIC TABLE
ACID 1
DETERMINATION OF AsA IN ASCORBIC ACID SOLUTIONS
PURE
AsA found (mg1100 ml) AsA added (mg1100 ml) 12.1 17.6 19.2 22.0
CBQ method 11.95 17.60 19.35 22.05
DCPIP method
-c 0.05 + 0.00 k 0.15 2 0.05
a Average of two determinations, ation from the mean.
12.10 17.50 19.15 22.25
pharmaceutical products is shown in Table 2. Most of the substances tested for interference, even when present in a concentra-
OF ASCORBIC ACID BY THE CBQ METHOD IN THE PRESENCE OF MINERALS, VITAMINS, REDUCING SUGARS, AND COMMON REDUCING AGENTS
Substance
MgSO, ZnSO,.7H,O Co(N0&.6H20 K,SO, MnSO, 3H,O Thiamine. HCl Nicotinamide Riboflavin Nicotinic acid Pantothenic acid Pyridoxine Fructose Glucose Lactose D-Mannose Sucrose Hydroxylamine-HCl Hydrazine-H,SO, Urea Thiourea Cysteine Glutathione Sodium thiosulfate FeSOI.7H,0 a Average of two determinations,
0.10 0.05 0.15 0.05
with average devi-
TABLE DETERMINATION OTHER
-t k -+ -t
Amount added (mg)
AsA added (ms)
1000 1000 1000 1000 1000 850 1000 10 1000 1000 1000 1000 1000 1000 1000 1000 200 100 1000 10 10 10 10 10
10.1 10.1 10.1 10.1 10.1 10.2 10.2 10.2 10.2 10.2 10.2 10.1 10.1 10.1 10.1 10.1 10.1 10.1 10.1 10.1 10.1 10.1 10.1 10.1
with average deviation from the mean.
AsA found” (mg) 10.15 10.15 10.00 10.20 10.10 10.25 10.20 10.15 10.10 10.20 10.20 10.05 10.05 10.10 10.20 10.10 10.30 10.05 10.00 10.05 15.20 12.70 13.05 13.05
t 0.05 f 0.05 2 0.00 k 0.10 t 0.10 2 0.05 f 0.10 k 0.05 + 0.00 -r- 0.10 + 0.10 2 0.05 + 0.05 * 0.10 r 0.00 f 0.10 k 0.05 +- 0.05 2 0.00 r 0.05 c 0.05 f 0.05 k 0.05 + 0.05
4
SHIEH AND SWEET TABLE
3
DETERMINATIONOFASCORBICACIDINTHEPRESENCE OF Fe*+ WITH AND WITHOUTDOWEX 5OW-X12 TREATMENT
FeSO,.7H,O added (md 40.0 40.0 40.0 40.0 0.0
AsA added (mg)
Dowex SOW-Xl2 added (Id
0.0 0.0 0.0 9.5 9.2
0.00 1.00 2.00 2.00 2.00
AsA found (mg)” 12.8 3.3 0.0 9.4 9.0
a Average of two determinations, ation from the mean.
+- 0.1 -c 0.0 c 0.0 2 0.2 5 0.1
with average devi-
tion as high as lOO-fold that of ascorbic acid, do not cause any interference. Either riboflavin or thiourea will give higher absorbance values ifpresent in more than an equal amount of ascorbic acid, but the quantities normally encountered for both of these substances are usually lower than this limit. Hydroxylamine-HCl may be present in quantities up to 20-fold that of ascorbic acid and hydrazine-H&SO, may be present in quantities up to IO-fold that of ascorbic acid without interference. The substances found to interfere in the analysis of ascorbic acid by the proposed method include sodium thiosulfate, ferrous sulfate, and sulfhydryl compounds such as TABLE
4
DETERMINATION OF ASCORBIC PHARMACEUTICALPRODUCTS
Tablet No.
AsA claimed (mg)
I II III IV V
250 250 300 60 60
ACID
IN
AsA found (mg)
CBQ 247.4 248.0 316.4 59.2 53.2
r + k + 2
n Average of three determinations ation from the mean.
DCPIP 0.6 0.8 0.4 0.2 0.2
244.0 246.6 318.4 60.5 54.2
f 1.4 ZII 0.8 + 0.8 2 0.5 + 0.6
with average devi-
cysteine and glutathione. These substances also interfere with the 2,6-dichlorophenol indophenol method (10). Van Eekelen and Emmerie removed sulfhydryl compounds and thiosulfate with mercuric acetate prior to analysis for ascorbic acid with 2,6dichlorophenol indophenol(l1). These substances are precipitated by mercuric acetate whereas the ascorbic acid remains in solution as dehydroascorbic acid. To remove the excess mercuric acetate after the precipitation and to reduce the dehydroascorbit acid back to ascorbic acid, H,S was used. Formaldehyde condensation was recommended by Lugg and Mapson to differentiate ascorbic acid from sulfite, thiosulfate, sufhydryl compounds, reductones, and reductic acid (12,13). In the present work it was found that the interference of ferrous sulfate, which is frequently present in pharmaceutical products, can be prevented by the use of the cationexchange resin Dowex 5OW-X12. For the analysis of Fez+-containing samples, 2 g of Dowex 5OW-X12 in the H+ form is sufficient to remove the iron in as much as 40 mg of FeSO, .7H,O present in ascorbic acid samples. Results are shown in Table 3. The proposed method has been applied to the determination of ascorbic acid in pharmaceutical products. Table 4 shows the results obtained for the analysis of the ascorbic acid content in five commercial vitamin products. Tablets I and II are vitamin C tablets. Sample III is a multivitamin capsule. Tablets IV and V are multivitamin preparations that also contain ferrous salt. Each sample solution of tablets IV and V was treated with the cation-exchange resin prior to analysis. REFERENCES 1. Hoste, J., Eeckhout, J., and Gillis, J. (1953)Anal. Chim.
Acta
9, 263-274.
2. Guest, J. (1953) Anal. Chem. 25, 1484- 1486. 3. Ptlaum, R. T., Popov, A. L., and Goodspeed, N. C. (1955) Anal. Chem. 27, 253-255.
SPECTROPHOTOMETRIC
DETERMINATION
4. Bahr, H., and Lipinska, I. (1968) Gem. Anal. (Warsaw) 13(2), 399-403. 5. Faye, G. H. (1966) Canad. J. Chem. 44, 27292736. 6. Taqui Khan, M. M., and Martell, A. E. (1967) J. Amer. Chem. Sot. 89, 4176-4185. 7. Bessey, 0. A. (1938)J. Biol. Chem. 126,771-784. 8. Morell, S. A. (1941) Anal. Chem. 13, 793-794. 9. LoeffIer, H. J., and Ponting, J. D. (1942) Anal. Chem. 14, 846-849.
OF ASCORBIC
5
ACID
IO. Sebrell, W. H., and Harris, R. S. (1967) The Vitamins, 2nd ed., Vol. 1, pp. 345-352, Academic Press, New York. 11. Van Eekelen, M., and Emmerie, A. (1936) Biothem.
J. 30, 25-27.
12. Lug, J. W. H. (1942) 20, 273-285. 13. Mapson, L. W. (1943)J. 62, 223-232.
Ausr.
Sot.
J. Exp.
Chem.
Ind.
Biol.
Med.
(London)
