V O L U M E 21, NO. 8, A U G U S T 1 9 4 9
1009
Perkin, H. J., Brown, B. R., and Lang, J., Can. M e d . Assoc. J . , 3 1 , 365 (1934). (40) Rabourdin, C o m p f . rend., 31, 784 (1850). (41) Rapp., E., Arch. Pharmag., 241. 328 (1903). (42) Homijn, 2. anal. Chem., 36, 18 (1897); 3 9 , 60 (1900). (43) Saifer, A . , and Hughes, J., J . Biol. Chem., 118, 241 (1937). (44) Salter, W. T., “Endocrine Function of Iodine,” pp. 270-91, Cambridge, hfass., Harvard University Press, 1940. (45) Sohezow. Z . anal. Chem.. 44. 86 (1905). (46) Sendroy, Julius, Jr.. and Alving, A. S., J . Biol. Chem., 142, (39)
159-70 ( 1 9 4 2 ) .
147) Sheiill, M. S.,Z . physak. Chem., 4 7 , 104 (1904).
(48)
Strickler, H. S., and Strickler, E. W., Endocrinology, 37, 220-2
(1945). 149) Sy, A. P., J . Am. Chem. Sac., 29, 786 ( 1 9 0 7 ) .
(50) Taurog. A., and Chaikoff, I. L., J . Bid. Chem., 163, 313-22 (1946).
Valeur, Bull. soc. chim., 23, 58 (1900). (52) Votecek, E., Chem. Ztg., 42, 257, 271, 317 (1918). (53) Winterstein and Herzfeld, Z . physiol. Chem., 63, 49 (1909). (51)
RECEIVED J u n e 9, 1948. P a r t of a dissertation presented by John J. Custer t o t h e Graduate Faculty of Brooklyn College in partla1 fulfillment of t h e requirements for t h e MS. degree.
Microdetermination of Riboflavin by Synthetic Ion Exchange Resin >IOTONORI FUJITdR.4 AND HIROSHI SHIhlIZU Icy0 to I;‘ni cersi ty , Kyoto, Japan h new method of chemical determination of riboflavin by using synthetic cation exchange resin (KH-9) has been studied, and a rapid and accurate microdetermination of riboflavin has been achieved with successful removal of nonriboflavin fluorescence.
N
UMEROUS physicochemical methods have been presented for the determination of riboflavin, all of which may be
classified as (1) direct measurement of color of riboflavin solution ( 7 , 8) or (2) measurement of fluorescence of riboflavin solution (3, IO). The latter method is more sensitive than the former. Adsorption I n applying the fluorescence method for the quantitative R-so~-.IIs+ / + determination of riboflavin in biological materials, it is necesElutiorl sary to remove as completely as R-SOB-.V-SH” HO possible fluorescing substances, which interfere with the accurate determination of tlir yellowgreen fluorescence due to riboflavin. For this purpose, the following method has been geiierally employed. Riboflavin is adsorbed on adsorbents of the fuller’s earth type, then eluted from such adsorbents with pyridineacetic acid or pyridine-alcohol solution, and the eluate is treateJ with potassium permanganate and hydrogen peroxide prior to measuring the fluorescence. But very few have adopted a “dynamic” method, which is more effective than a static method for adsorption and elution. In 1911 Conner (2) proposed adsorbing and eluting riboflavin by a dynamic method, using Supersorb, one of the fuller’s earth group. I n his combined determination of riboflavin and thiamine, he adsorbed riboflavin on Supersorb and thiamine on zeolite, and eluted riboflavin by pyridine-acetic acid solution and thiamiiie by potassium chloride. This method is coiiipnratively cainplicnteJ because the operation must employ a vacuuni system with special apparatus. For this reason, the authors studied n new addorbent of riboflavin by which. the operation can be carried out under n o r i n d pressure with a simple apparatus, and found that the synthetic cation exchange resin KH-9 is most suitable for this purposc.
+
V-S HzO +V=XH+.OHTherefore riboflavin can be adsorbed on a cation exchange resin by a cationic exchange reaction and eluted with pyridine as follo\rs :
c,... +
PRINCIPLE OF THE METHOD
Adsorption and Elution. The structuie of ribofla-b.iri, in reference to the basic nitrogen, may be repreaented as: \-=S. Accordingly, in aqueous solutioii it undergoes dissociation as follolvs:
The cation exc!iange resin can be used repeatedly, as shown in Equations 1 and 2. Removal of Nonriboflavin Fluorescence. Most of the interfering substances possessing fluorescence may be removed by the following procedures. The nonriboflavin fluorescent substances in the extract, which have a stronger adsorption affinity than riboflavin, should be adsorbed on pyridine-treated zeolite, and those with an adsorption affinity equal to or less than riboflavin should be adsorbed on a synthetic resin. Then nonriboflavin fluorescent substances with a weaker affinity than riboflavin should be removed by rinsing the resin with hot water. The remaining nonriboflavin fluorescent substances with an equal adsorption affinity to riboflavin should be eluted with pyridine-acetic acid solution, and interfering substarices possessing fluoresce:ice should be reduced by oxidation with potassium perniangariate solution and then by the use of a suitable yelloi7 filter, the maximal transmission of which should be 560 mp. PRACTICAL PROCEDURES
Reagents. Synthetic Cation Exchange Resin KH-9. The Japanese Vitamin Pharmacal Co., Osaka, or the Oda Laboratory of Kyoto University. Synthetic Zeolite. Takeda Cheniical Co. Pyridine-hcetic -kcid Solution (pH 7.0), 20 or 30 volume yo. Glacial ricetic Acid. Potassium Pcrmangmate Solution. A 4yc solution, freshly prepared each week. Hydrogen Peroxide Solution. .4 37, solution is prepared by diluting a 3Oor solution rtf hydropr.n pc3rouidr with distillrti water.
ANALYTICAL CHEMISTRY
1010
Standard Rihotlavin Solution. A 5 mg. % riboflavin solution is prepared with distilled water (adding S drop of glacial acetic acid to 100 ml. of water). It is diluted 1 to 50 with distilled water at the time of using. Takadiastase. A 2% takadiastase solution is prepaxd and used after filtration through both zeolite and resin column. Procedure. Two exchange tubes made of brown glass 0.7 to 0.8 em. in diameter, are prepared. Figure 1 represents two assemblies. The upper column is Bled with 1.5 grams of activated zeolite of 60-to SOSmesh, and the lower column with the same amount of purified KH-9 of the same mesh. The former is treated with 50 ml. of loyopyridine-acetic acid solution and then about 2 0 ml. of distilled water. The latter is ,treated with sufficient hot water and about 50 volume 70pyridine solution until the resin gives no fluorescent substances in the eluate of 20 or 30 volume yo pyridineacetic acid solution, and is then rinsed with 200 ml. of distilled water. The eluate is preserved as a blank solution. A finely pulverized sample, containing from 2 to 5 micrograms of riboflavin, is weighed, and about 40 ml. of distilled water &PO added. The pH of this mixture iS adjusted to 4.5 with 1N hydroohloric acid or sodium hydroxide. After addition of about 2 ml. of takadiastase solution and a few drops of toluene, the mixture is' allowed to stand overnight in an incubator a t 38' C. Then the mixture is heated on a boiling water bath for 15 minutes, being continuously stirred, after addition of 5 ml. of 1 N sulfuric acid. The extract is cooled to room temperature, d i e tilled water is added to make the total volume of liquid 50 ml. and i t is centrifuged at)high speed until a clear supernatant liquid is obtained. The extract, which contains proteins, must he treated to precipitate with just enough 10% metaphosphoric acid. A certain &mount of clear supernatant solution, containing from 1 to 3 micrograms of rib0 flavin, is allowed to pas through the two column at the rate of 1 to 2 ml per minute, after pH ha been adjusted to 4 to ! with sodium hydroxide Both columnsare washe( down thoroughly 6 t o ! times with 5-ml. volume, of distilled water succes
sorption affinity- thai riboflavin are adswbu on the pyridine-treate( zeolite, but those with ai adsorption affinity equa to or less than ribotlavii pass through it and an adsorbed on the lowe column of KH-9. The resin oolumn ii
Table I. 'Riboflavin C o n t e n t of Biological M a t e r i a l s Ribo &"in
Material
Riboflavin content,
Pumpkin potato
Watermelo, Tomato
~~~
~
~
~~
measurid in oomparison with the fluorcscenco of the riboflavin solution obtained by the titration of the blank test solution with standard riboflavin solution, by using a suitable yellow filter. ANALYTICAL RESUL'Irs
T he riboflavin content of various biologic:a1 materials, analyzed by t h is method, is presented in Tahle I. DISCUSSION
KH-9 is a synthetic resin with p - and o-phenolsulfonic acids s,s its base (9), which bas a large ion exchange capacity and is very stable physicochemically. The break-through capacity is above 500 micrograms per gram of the resin. It can be used more than 200 times repeatedly in these determinations. The adsorption of riboflavin on KH-9 under the conditions described above is always complete. But as an organic solvent interferes with adsorption, an extraction employing alcohol or acetone must he avoided. Canner ( 8 ) reoognieed t h a t the aeolite. has a weak affinity for riboflavin. The authors have found that the pyridine-treated zeolite has less affinity for rihoflavin than the potassium seolite, and t h a t the riboflavin adsorbed on pyridine-treated zeolite is eluted easily by a little distilled water without any leakage of thiamine. The authom' experiments show t h a t 4 to 10 micrograms of riboflavin in 10 ml. of distilled water adsorbed on 1.5 grams of pyridine-treated zeolite are completely eluted by ahout, 3:Oml. of distilled water. As the eluates ohtsined by this method always give yellow:reen fluorescenoes due to riboflavin, more accurate results may e obtained by using a fluorometer, as generally employed in t h e I:N IJnited States for the determination. _. . . . . . . . . 'The recovery at the pure rlbomvln SOIUtlOn added to the ext mct WBS always more than 90% (Tahle 11).
Table 11. fluorescence id fouud ir the solution pasre( through. Then ribn flavin and the other suh. stances with rn adsom tion affinity equal i( riboflavin me e l u k d wit1 25 ml. of 20 or 30 volume % pyridineacetic acic solution at a rate of 1 ml. per rmnnte. The eluate IS made u p to exactly 25 ml. in a graduated c y h der After mxine. 2 to 4 ml. of the elu& are pipetted into a test tube
Con tent,
Material Rice, brown Rice bran Soybean Kidney bean (fresh) Perilla Leaf of bee Leaves of J Turnip grei Japanese TB
Peroentage Reoovery of Added Riboflavin
Salnple
Riboflavin Content of Sample
RiboHsvin Added
Re Rib
7/#.
7
Y
R:ice bran
5.04
T omato
2.50 0.39 0.22
1.0 1.0 2.0 2.0
0.97 0.95
L;e&ves of Jspanese radish
I.[cine
1.84 1.96
97 95 92 98
As thiamine is usually analyzed by using pyridine-treated zeolite ( 4 , 5 ) ,a combined determination of t,hiamine and riboflavin can easily he achieved by this method. Herr (6) and Brown ( 1 ) found that Amberlite I R S O O adsorbs riboflavin but they have not yet applied this resin to the determination. The authors have found that KH-9 is suitable not
V O L U M E 21, NO. 8, A U G U S T 1 9 4 9
1011
only for the determination of riboflavin but also for the determination of pyridoxine and nicotinic acid by the dynamic method. 4CKNOWLEDG.MENT
The authors wish to thank U. Miura, R. Oda, and M. Nishio of Kyoto University for valuahle advice and assistance in this investigation. LITERATURE CITED
1\11 Brown, E. B., Bina, A. F.. and Thomas. T. &JI..Biol. . Chem , 158,455 (1945).
(2) Conner, R. T., and Straub, G. J., IKD.ENG.CHEY.,ASAL. ED., 13,385 (1941). (3) Euler, H. von, and Adler, E., Z . physiol. Chem., 223, 105 (1934). (4) Fujiwara, M., and Kitamura, S., J a p a n . J . Hug., 2, 12 (1948). ( 5 ) Hennessy, D. J., IND.Esc;. CHEM.,ASAL. ED.,13, 216 (1941). ( 6 ) Herr, D. S., I n d . Eng. Chem., 37, 631 (1945). (7) Kharit, 9.Yu., and Khaustov, N. W.,Biochem. J . , 29, 34 (1935) (8) Koshara, IT., 2. physiol. Chem., 232, 101 (1935). (9) Ode, R., Shirnizu, H., and Nakayama, Y . , Chemistry of High Polymer, to be published. (10) Supplee, G. C., Ansbacher, S., Flanigan. G. E., and Hanfod. Z . M., J . Dairy Sei., 19, 215 (1936).
K E C E I V F . ~September 22.
1948.
Volumetric Determination of Small Amounts of Iron Chromous Chloride as Reducing Agent WILLIAM D. COOKE, FRED HAZEL, AND WALLACE RZ.
3ICNABB
University of Pennsylvania, Philadelphia, Pa.
communication showed that chromous chloride Lamalgam 4 PREVIOUS gave excellent results when employed with liquid zinc as a reducing agent for ferric ions (f and that its favor),
Table I. Determination of Iron Fe Taken,
Fe Found,
hlg.
A1g.
%
9,047
9.022 9.013 9.026 9.018 9.020
-0 3
Mean 3.619
Error,
-0.4 -0.2 -0.3
3.632 3.626 3.615 3.613 Mean 3.621
f0.3 +0.2
1.805
-0.3 f0.6
1.810
1.820 1.810
-0.1 -0.2
0 -0.1
Mean
1.808 1.811
+0.8
Mean
0.730 0,724 0.717 0.713 0.721
0,362
0.358 0.357 0.357 0.363 Mean 0.359
-1.1 -1.4 -1.4 $0.3
0.302
0.301 0.295 0.301 0.294 0.298
-0.3
0.119 0.120 0.119
-0.8
0.724
Mean 0.120
0.120
Mean 0.120
0 -1.0 -1.5
-2.3 -0.3 -2.6
0
-0.8 0
able application as a reducing agent depended upon the fact that the excess chromous ion is oxidized by atmospheric oxygen with no appreciable oxidation of the iron under the conditions of the experiment. Phenosafranine, a low potential redox indicator, was used to follow the reactions. Chromous chloride alone is a satisfactory reducing agent for small amounts of iron. The method can be applied to solutions containing 0.1 mg. to 10 mg. of iron satisfactorily; viith larger amounts of iron, the green color of the chromic ions interferes. A single determination can be carried out in 2 to 3 minutes n-ithout the use of an inert atmosphere. The reagents were used a t the concentrations shown in the following procedure and viere prepared by methods already described ( 1 ) . The average error for twelve determinations using between 1 and 10 mg. of iron was 0.250J0. The average error was 0.9% in sixteen determinations using less than 1 mg. of iron. Five to 25 ml. of ferric iron solution containing 0.1 to 9 mg. of iron are acidified with either sulfuric or hydrochloric acid, and 2 or 3 drops of 0.017, phenosafranine indicator are added. Chromous chloride solution is then added dropwise until the pink color of the indicator disappears and the solution becomes a light clear green. The solution is swirled until the pink color reappears (this color is not the same as the original pink because of the added chromic ions). A few drops of phosphoric acid and 0.05 to 0.1 ml. of a 0.16% diphenylamine sulfonate solution are added and the ferrous iron is titrated with a solution of potassium dichromate. A 0.007 i Y solution of potassium dichromate is used for amounts of iron greater than 1 mg. A 0.003 S solution is used for less than 1 mg. The end point is a purple color which is easily detected over the light pink or violet color of the solution. A correction of 0.05 ml. of 0.01 ?; potassium dichromate is subtracted for each 0.1 ml. of indicator used in the titration. In the titration of less than 1 mg. a solution of the oxidized form of the diphenylamine sulfonate was used and gave a blank of 0.01 ml. of 0.01 iz’ potassium dichromate for each 0.1 ml. of indicator. LITERATURE CITED
(1) Cooke, Hazel, and McNabb, ANAL.CHEM.,21, 643 (1949). RECEIVED November 19, 1948. Presented before the Meeting-in-Miniature. Philadelphia Section. AYERICAX CHEMICAL SOCIETY, January 20, 1949.
