Estimation of Vitamin
C
in Presence of Iron Salts Vitamin C and Ferrous Iron with
Stepwise Determination of Dichlorophenolindophenol OSCAR GAWRON AND RUTH BERG
Research Laboratory, International Vitamin Division, American Home Products Corp., N e w York,
6% Metaphosphoric Acid, 60 grams of crushed metaphosphoric acid sticks dissolved in distilled water and made up to liter. When not in use this solution was kept in the refrigerator. Citrate and Metaphosphoric Acid Buffer, as described by Bessey ( 1 ) Dichlorophenolindophenol Dye Solution, 50 mg. of etherextracted dichlorophenolindophenol dissolved in 200 ml. of d i 5 tilled water to which 42 mg. of sodium bicarbonate had been added. When not in use the dye was stored in the refrigerator.
R
EPORTS on the reductive interference of ferrous iron with the dichlorophenolindophenol reagent used in the estimation of vitamin C have appeared in the literature and procedures have been devised to minimize and remove this interference (2-5). During the course of studies in this laboratory on isolated vitamin C-iron salt systems the authors have found that ferrous and ferric iron interferences can be avoided if a suitable medium is chosen for the dye titration. Thus vitamin C can be determined in the presence of ferrous iron if acetic acid is employed as the titration medium and in the presence of ferric iron if metaphosphoric acid is present in the medium. The observation that ferrous iron reduces dichlorophenolindophenol in the presence of metaphosphoric acid provides a basis for the stepwise determination of vitamin C and ferrous iron in the same aliquot. Whether or not these observations may be applied in toto or in part to biological systems, pharmaceuticals, and food products where other interferences (3) in addition to iron may be present remains to be determined.
Table
Estimation of Vitamin
C and Ferrous Iron
Total Dyea Subsequent addition of 20 ml. 10 ml. of 6 % metaof 8% phosphoric acetic acid acid
MZ.
M1.
Found Vitamin Ferrous C iron Mg. Mg. 0.0 0.63
0.64 mg. of ferrous iron 0.05 9.1 1.0 mg. of vitamin C 9.2 9.2 0.99 0.00 1.0 mg. of vitamin C and 0.64 mg. of ferrous iron 9.3 18.6 1.01 0.64 Vitamin C b capsule containing FeSO4 12.8 21.6 34.7 15.1 Dye standardized against U.S.P.vitamin C. b Vitamin capsule containing 35 mg. of vitamin C and 15 mg. of iron diluted t o 25 ml., 1-ml. aliquot used for titration. Capsule found by oxidai tion and colorimetric estimation with Lhiocyanate to contain 15.0 mg. of iron
REAGENTS
1.
111.
Sample
Ferrous Iron Solution. Two grams of dried ferrous sulfate powder were dissolved in 160 ml. of distilled water containing 5 ml. of concentrated sulfuric acid by warming gently on the steam bath. The solution was treated with a rapid current of hydrogen sulfide for 0.5 hour, followed by a stream of nitrogen, and then made up to a volume of 1000 ml. with distilled water. An aliquot titrated with 0.01 N potassium permanganate contained 0.64 mg. of ferrous iron per ml. 8% ilcetic Acid, 80 ml. of glacial acetic acid made up to liter with distilled water. Table
N. Y.
EXPERIMENTAL AND DISCUSSION
.Table I shows that ferrous iron reduces dichlorophenolindophenol in the presence of metaphosphoric acid. This also happens when phosphoric acid is presgnt' and can be explained on the basis of the increase in reduction potential of the ferrousferric system when the effective ferric-ion concentration is reduced by complex formation with metaphosphate or phosphate ions. The reverse of the above phenomena takes place in the presence of ferric iron. Here, as can be seen from Table 11, the reduced dye is reoxiciized in acetic acid medium but not in the presence of metaphosphoric acid. This reoxida tion takes place a t a much slower rate than the corresponding reduction by ferrous iron and at the present time is being investigated ns the basis for a colorimetric estimation of iron. From the above experiments it was apparent that vitamin C and ferrous iron could be determined stepwise on one aliquot by first titrating in acetic acid, then adding metaphosphoric acid and continuing the titration. Control analyses and the analysis of a vitamin capsule containing vitamin C and ferrous sulfate are given in Table 111.
Influence of Medium on Reduction of Dichlorophenolindophenol by Ferrous Iron
(0.64 mg. of ferrous iron present) Dichlorophenolindophenol Required for E n d Point, Titration Medium= M1. 8% acetic acid 0.05 3 % metaphosphoric acid 9.1 6% metaphosphoric acid 9.2 Citrate reagent (Bessey) Slowly fading end point 8% acetic acid adjusted t o p H 3.5 with NaOH 0.05 8% acetic acid with 0.0257, metaphosphoric acid present 3.2 Reduction very slow and not reproducible 8% acetic acid with 0.15% metaphosphoric acid preaent 5.0 8% acetic acid with 0.5% metaDhosuhoric . acid present 7.3 8% acetic acid with 1.0% metaphosphoric acid present 9.0 8% acetic acid with 3% metaphosphoric acid present 9.1 Initial volume 25.0 ml.
SUMMARY
Ferrous sulfate reduces dichlorophenolindophenol in the presence of metaphosphoric acid. Ferric iron oxidizes reduced dichlorophenolindophenol in acetic acid medium. Vitamin C and ferrous iron can be determined on one sample by stepwise titration with dichlorophenolindophenol.
Table II. Influonce of Medium on Titration of Vitamin C with Dichlorophenolindophenol in Presence of Ferric Iron (1.00 mg. of vitamin C and 0.75 mlc. - of ferric irona Dresent in all cases) Dichlorophenolindophenol Required for E n d Point, Titration Mediumb M1. 8% acetic acid 5.9= 8 % acetic acid with 3% metaphosphoric acid present 9.2 3% metaphosphoric acid 9.3 6 % metaphosphoric acid 9.2 From ferric ammonium sulfate Standardized by reduction and subse uent titration with KMnOd. Initial volume 25.0 ml. C Not reproducible.
LITERATURE CITED (1) Bessey, 0. A , , J . B i d . Chem., 126, 771 (1938). (2) Harris, L. J., and Ollivcr, M., Biochem. J., 36, 155 (1942). (3) Hochberg, M., Melnick, D., and Oser, B. L., IND. ENG.CHEM.. ANAL.ED.,15, 182 (1943). (4) Singer, J. H., and Milner, M . N., Analyst, 68, 272 (1943). (5) Woessner, W. E., Elvehjem, C . A., and Schuette, J. A,, J . Nutrition, 20, 327 (1940).
?I
751
