INDUSTRIAL AND ENGINEERING CHEMISTRY
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whereas the concentration of these substances in the solution treated with sulfite may now be below this range. If the treated solution is below an equivalent of 10 millimicrograms, it is necessary to add 10 millimicrograms of thiamine ( 1 ml. of thiamine standard solution A) before making up to volume. Experience has shown this to be necessary generally with food samples but not with urine samples.
Fermentation Procedure Prior to each run, grease all ground joints, open the capillary gas valves to the cups, and set the water bath to 30" C. I n the main chamber of each reaction flask place 1 ml. of distilled water. In the inset place 1 ml. of the test solution or suspension which contains carbon dioxide-stimulating substances equivalent to 10 to 20 millimicrograms of thiamine, or 1ml. of thiamine standard solution A or B. Then prepare the buffer-substrate-yeast suspension by mixing in a 100-ml. volumetric flask 15ml. of solution B, 10 ml. of solution A, and 7 ml. of 15 per cent ammonium sulfate followed by 25 ml. of 2 per cent yeast suspension. Elapsed time is measured from the time the yeast suspension is added. Make the buffer-substrate-yeast suspension to volume and add 1 ml. to the inset of each flask. Fix the flasks to the manometers, place in the water bath, and flush with nitrogen through a manifold. Ten minutes have usually elapsed a t this point if 12 Summerson units (24 Warburg units) are employed. Shake the flasks at 140 oscillations (70 round trips) per minute a t an amplitude of 3 cm. Stop the passage of nitrogen after 50 minutes have elapsed, and close the capillary gas valves to the cups. Release excess pressure in each unit 5 minutes later. Make a zero reading at exactly 60 minutes, close the system, and make a final reading 60 minutes later. Calculations are based on the comparison of gas evolved in the test solution after correcting for nonthiamine stimulating substances with the gas evolution due to standard thiamine solutions A and B, assuming a linear relationship between thiamine concentration and carbon dioxide evolution over the 10 to 20 millimicrogram range. The gas evolution due to standard thiamine solutions A and B must be determined at each run.
Kith this modified procedure repeated estimations of the thiamine content of various plant and animal tissues and fluids have shown an agreement within 2 per cent (Table I).
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Furthermore, these results agree within 5 per cent with those obtained by fluorometric assays of the same samples (Table I). This fermentation procedure is preferred to fluorometric methods in the analysis of samples containing very small quantities of thiamine or when information is desired on the fate of thiamine during the handling and preparation of foods and in tissue metabolism. Over 1000 successful thiamine determinations have been performed by the authors using the method described.
Summary A revised procedure for the microestimation of thiamine in tissues and tissue fluids using the Warburg technique is presented. Interference by nonthiamine substances is corrected by sulfite treatment. Residual sulfite is eliminated by a peroxide treatment. This procedure is especially applicable to the assay of tissues and fluids low in thiamine (10-8 gram of thiamine per ml.) and in the measurement of thiamine degradation products in tissue metabolism and in the treatment of foods.
Acknowledgment Technical assistance from Elizabeth Weeks and Helen C. Scrufutis is acknowledged.
Literature Cited (1)
Atkin, L., Schultz, A. S., and Frey, C. N., J. Bid. Cheni., 129,
(2)
Schulta, A. S.,Atkin, L., and Frey, C. N., IND. ENG.CHEM.,
471 (1939). ANAL.ED., 14, 35 (1942). (3) Summerson, W. H., J. Bid. Chern., 131, 579 (1939). (4) U. S. Pharmacopoeia X I , 2nd Supplement, p. 131, 1939. FROMt h e Department of Biology and Biological Engineering, Massachusetts Institute of Technology, Cambridge, Mass. T h i s research has been supported hy a grant from the Williams-Waterman Fund of Research Corporation.
Colorimetric Microdetermination of Iron C. P. SIDERIS, Pineapple Research Institute of Hawaii, Honolulu, T. H.
T
HE observation of van Klooster (1) in 1921 that nitroso R salt forms a green color with ferrous iron was later con-
firmed by the writer (2). Further studies on the reaction between ferrous iron and nitroso R salt, started in connection with various potassium analyses where iron occurred as a contaminant, suggested the possibility of using this reagent for the determination of ferrous iron, The method, as described below, has been found satisfactory for determining iron in plant tissues.
Reagents STAXDARD IROXSOLUTIOK.Dissolve over a hot late 1 gram of metallic iron C'rust-free" wire) in a 1000-ml. flasi containin 10 ml. of concentrated sulfuric acid and 100 ml. of water, coo$ and make to volume with water. Standards containing 0.2 t o 20.0 micrograms of iron per ml. are prepared from this solution. XITROSO R SALT, Place 0.5 gram of nitroso R salt in a 100-ml. volumetric flask containing 70 ml. of water, dissolve by gentle agitation, and then complete the volume with redistilled (ironfree) acetone. The reagent keeps well for many months. SODIUM ACETATE. Place 544.3 grams of sodium acetate (ironfree) in a 1000-ml. volumetric flask, then pour in 300 ml. of water, and shake gently at intervals while heating on a hot plate until complete solution, Make to volume with water and filter.
HYDROXYLAMINE SULFATE.Place 10 grams of hydroxylamine sulfate in a 100-ml. volumetric flask containing 80 ml. of water, shake until complete solution, and complete the volume with water.
Procedure Ash about 5 grams of dry plant tissue in a platinum crucible. Dissolve the ash with 10 ml. of 5 N hydrochloric acid by heating slowly over a hot plate. Add to the platinum crucible approximately 20 ml. of water. Heat the mixture to about 90" C., transfer to a 100-ml. flask, cool nearly to room temperature, dilute to the mark, and filter. Place a 10-ml. aliquot of the solution in a test tube and neutralize with 5 N sodium hydroxide in the presence of a very small piece of litmus paper. Remove the latter with a glass rod and add 1 ml. of hydroxylamine sulfate, 1 ml. of nitroso R salt, and 2 ml. of sodium acetate. (Either 2 ml. of sodium acetate or 1 ml. of 1.5 N ammonium hydroxide can be used for adjusting the solution to pH 8 t o 10.) Make to 15 ml. in a graduated test tube and allow to stand for 20 mlnutes or longer. Transfer to a cell, preferably 2.5 mm. in thickness, and determine the color intensity with a photoelectric colorimeter, using a No. 47 light filter with transmission limits 445 to 505 millimicrons. A 2.5-mm. cell can be obtained by using a plunger 7.5 mm. thick in a 10-mm. cell as supplied with the Summerson-Klett photoelectric colorimeters. In case of formation of a precipitate by calcium with sulfate or
ANALYTICAL EDITION
September 15, 1942 ~~~~
Ferrous iron forms with nitroso R salt (1-nitroso- 2- hydroxy 3,6-naphthalene disodium sulfonate) at favorable range of pH values (8 to 10) a green pigment which is in direct proportion to the amounts of ferrous iron present in the solution. The sensitivity of the reagent is very great, reaching the low limits of 0.2 microgram of ferrous iron per ml. and ranging between 0.2 and 50 micrograms or more. Hydroxylamine sulfate was found highly satisfactory for the reduction of ferric to ferrous iron and sodium acetate or ammonium hydroxide for obtaining a satisfactory pH range for the development of the green pigment. Iron may be determined by means of a photoelectric colorimeter with the use of appropriate light filters.
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phosphate ions after the addition of sodium acetate, centrifuge, decant the supernatant liquid, and discard the precipitate. Turbid solutions interfere considerabIy with the accuracy of the method.
Results Table I and Figure 1 present values obtained with a Summerson-Klett photoelectric colorimeter for different concentrations of iron with three different amounts of nitroso R salt. The data indicate t h a t 0.5 ml. of nitroso R salt, as reported in Table I, was sufficient only for quantities of iron ranging from 0.25 to 10.0 micrograms; and that 1.0 ml. of nitroso R salt covered twice as great a range of ferrous iron concentrations-that is, from 0.25 to 20 micrograms. With a volume of 2.0 ml. of nitroso R salt a range of iron concentrations from 0.25 to 25.0 micrograms was covered satisfactorily. A greater extension of the range from 25.0 to 30.0 micrograms of iron with 2.0 ml. of nitroso R salt was possible but with an error ranging from 2 to 10 per cent, resulting probably from the decreased sensitivity of the photoelectric colorimeter a t the higher regions of its logarithmic scale. The range of iron concentrations from 0.25 to 20 micrograms is the most satisfactory for photometric or colorimetric determinations.
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With considerably greater concentrations of ferrous iron in the unknown than 20 micrograms the writer recommends dilution and proper adjustment of the volume of nitroso R salt. Aqueous solutions of nitroso R salt are yellow. I n the presence of ferrous iron they form a green color, the amounts of which are directly proportional to the quantities of iron. Because of the existence of a stoichiometric relationship between ferrous iron and nitroso R salt, as shown in Table I, it is desirable to add nitroso R salt to solutions employed for the determination of iron in quantities directly proportional to those of iron. The stoichiometric relationship is evidenced by the fact that approximately 2.5 and 5.0 mg. of nitroso R salt, contained in 0.5and 1.0 ml., respectively, of 0.5 per cent solution were used up completely by solutions of ferrous iron corresponding in the former case with 0.1 mg. (10 ml. X 10 micrograms = 0.1 mg.) and in the latter, with 0.2 mg. (10 ml. X 20 micrograms = 0.2 mg.) of iron. The proportions of the reacting amounts of nitroso R salt and iron, based on the above data, were approximately 25 to 1. Comparing this with the ratio of the molecular weights of nitroso R salt (CloH&OsS2Sa2= 475) and iron (Fe = 55.84) which is 8.5 to 1 (475 -+ 55.84 = 8.5); the iron salt of l-nitroso-2-hydroxy-3,6-naphthalene disodium sulfonate has the formula Fe(C1OHSNOaS2Na2)3. The difference of 2 per cent between the obserT-ed ratio of 25 to 1 and the calculated ratio of 25.5 to 1 is relatively small and within the limits of an experimental error. Van Klooster (1) has suggested the formula (CloHJXOshTaz)3Co for the corresponding salt of cobalt. The addition of amounts of nitroso R salt greatly in excess COLORIWETER SCALE TABLEI. VALUESON PHOTOELECTRIC (For different amounts of iron per ml. of solution a n d nitroso R salt added) Nitroso R Salt Iron Y 0.5 mi. 1.0 ml. 2.0 ml. n 25 > 2 3 6 4 4 0.50 9 8 8 1.00 23 20 2.50 19 44 42 41 5.00 66 63 62 7.50, 85 82 10.00 78 10s 12.50 1 85U 106 124 127 1.5.00 865 145 142 17.50 870 165 20.00 160 88" 182" 184 22.50 88 0 182" 202 25.00 86" 240 184'' 30.00 87 L Results obtained with insufficient quantities of nitroso R salt as comDared with those of iron. ~~
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TABLE 11. COLORIMETER READIKGS (Obtained with a Sumrnerson-I
