V O L U M E 21, NO. 8, A U G U S T 1 9 4 9 1. The cells should be checked against the control cell. Any discrepancy in the reading should be noted and corrections made in the final reading. 2. It is of the utmost importance that just before each reading the 0 mark on t,he transmission density knob be checked against the distilled water control. For this purpose the switch pointer remains on 1.0, the transmission density is set a t 0, and the shutter is opened, allowing the light to pass through the cell containing distilled water. The adjustment is made with the sensitivity control and the dark current as required in the operation of the instrument. The reading of the unknown is made immediately after this adjustment. The spectrophotometer should be calibrated against standard iodate solutions in 0.1 N hydrochloric acid. Sext, 1.0 ml. of the standard is pipetted into the 4-ml. calibrated test tubes, the volume is brought to 4.0 ml. by the addition of redistilled water, 1.0 ml. of O.lyopotassium iodide solution is
lo05 added to each tube, and the reading is made according to the above-mentioned directions. The slightly higher concentration of hydrochloric acid does not influence the results in any way. LITERATURE CITED
L., ISD. ESG. CHEM.,i l w a ~ ED., . 12, ]KO (1940). (21 Custer. J. J., and "atelson, S., ANAL.CHEM.,21, 1003 (1949). ( 3 ) Fashena. G. J., and Trevarrow, V., J . Bid. Chem., 114, 351 i l l Charie), A .
(1936).
Leipert, T., Biochem. Z.,261, 437 (1933). ( 5 ) Matthews, N. L., Curtis, G. M., and Brode, W. R., IND. ENG CHEW.,ANAL.ED.,10, 612 (1938). (6) Shahrokh, B. K., J . BWZ. Chem., 154, 517 (1914). ( 7 ) Trevarrow, V., andFashena, G . J . , I b i d . , 110, 29 (1935). (4j
Hr-i
L1VF:D . r ' l l \ '
7 . 1918.
Spectrophotometric Determination of Microquantities of Iodine JOKY J. CUSTER AND SAMUEL NATELSOK Yrooklyn College and the Jewish Hospital of Brooklyn, Brooklyn, .V. Y Procedures are described for the spectrophotometric microdetermination of iodine. Light absorption spectra are reported for iodine in water, potassium iodide solutions, benzene, toluene, alcohol, and chloroform. The high absorption peaks in the ultraviolet region for elemental iodine in toluene, benzene, and potassium iodide solutions permit these solvents to be used for the determination of microquantities of iodine with the spectrophotometer. Sensitivity is increased sixfold by converting the iodine to iodate with alkaline permanganate. The iodate reacts with iodide in the presence of acid to liberate the iodine to be determined. Iodine is transferred by extraction procedures to the benzene, toluene, or potassium iodide solutions. 4 s little as 0.2 micregram of iodine is easill determined b) these methods.
T
H E need for a method, suitable for routine use for the determination of iodine in quantities on the order of 0.2 niicrogram as found in 1 nil. of human serum, has become of ii!creasing importance. This determination is not performed in most biological laboratories because of the elaborate procedures I ) I large quantities of blood serum required for a single determiriation. The authors' purpose was to devise a procedure which could be applied with reasonable precision and accuracy to the routine determination of microquantities of iodine. Thiosulfate titration of iodine is limited to the determination d a concentration of 7.5 micrograms of iodine per ml. (65). The usc of organic solvents such as benzene, petroleum ether, chlorotorni, and carbon tetrachloride as indicators in the titration of iodine has been proposed ( 7 , 40, 4 6 ) . These procedures increase the sensitivity of the titration so that 6.0 micrograms per ml. of iodine may be detected (25). A sensitivity of approximately 2 microgram of iodine per ml. in the presence of excess of iodide ion is claimed (66). .\rsenious oxide, trivalent antimony ( 2 6 ) , sulfurous acid ( 8 ) , hydrogen sulfide ( 6 4 ) , stannous ion, and thiocyanate ( 2 1 ) have been recommended for the titration of iodine. However, none of these appears to have a greater sensitivity for the'determination of minute quantities of iodine than thiosulfate. Organic compounds such as formaldehyde ( 4 2 ) , chloral hydrate ( 4 l ) , aldoses (S), acetone ( 1 4 , 36, 49), and hydroquinone ( 5 1 ) have also been suggested for this purpose. These methods have not suggested d procedure wherein the sensitivity would be great enough to dvtermine the quantities of iodine found in 1 ml. of blood serum. Titration methods, using adsorptinn indicntors, hnwd upon
the precipitation of insoluble iodides have also been proposed (10, 11, 22, 23, 28, 29, 37, 38, 47,52). The sensitivity of these methods is less than that for the thiosulfate titration. Electrometric titration of the reaction between iodine and thiosulfate (SO) was not found practicable for routine determinations of minute quantities of iodine. The methods wherein iodine is used as a catalyst for the reaction between ceric sulfate and nitrite (18)or arsenite (9, 48, 5 0 ) are capable of determining amounts of iodine in the required range. However, these catalytic methods are delicate, and require accurate timing, careful temperature control, and special apparatus. I n this laboratory, these methods were found to be time-consuming and not uniformly successful. In view of the chromophoric character of elemental iodine itself, it was felt that a colorimetric procedure might be developed with the use of the spectrophotometer. Various colorimetric methods for the determination of inorganic iodine have been proposed (1, 12, 13, 34, 58, 43, 46). These methods use the visible portion of the spectrum in reading iodine concentration. In the visible range the extinction coefficient for iodine is not high enough to be useful for minute quantities of iodine i n mater or other solvents ( 4 6 ) . Higher peaks have been reported for elemental iodine in potassium iodide solutions in the ultraviolet ( 4 , 4 6 ) . Because the state of iodine varies with the nature of the solvent, the authors decided to investigate the absorption spectra of iodine in several solvents to see whether a higher extinction coefficient could be found. This study was extended over the entire range nf the Beckman spmtrophotometer for the purpo.e
1006
ANALYTICAL CHEMISTRY
of finding a suitable peak. The solvents investigated included water, potassium iodide solutions, ethanol, chloroform, benzene, and toluene. The extinction coefficients are plotted in Figures 1 and 2 against the wave length. For extinction coefficients above 600 the graph in Figure 1 has been condensed to permit the demonstration of high peaks of iodine in potassium iodide solution. Figure 2 is drawn to a different scale to show the peaks for the poorly absorbing solvents. I t is apparent from an inspection of Figures 1 and 2 that potassium iodide solution would be the most suitable solvent. In order to avoid the need for a hydrogen lamp and to adapt the procedure for use Lvith other spectrophotometers, the peak at 352 mp was chosen. The effect of change in concentration of potassium iodide on the extinction coefficient was studied. Table I lists the extinction coefficients of three different concentrations of potassium iodide. I n each case the concentration employed was 2.13 micrograms of iodine per ml I t is evident from Table I that 5% potassium iodide iolutiori is perfectly suitable for this determination, for slight changes in potassium iodide concentration would not affect the extinction coefficient beyond the requirements of the method. Prior to the actual isolation of iodine in the potassium iodide solution it is necessary to destroy any organic material associated with the iodine. Two methods for the destruction of organic matter have been employed: alkaline fusion (15, 19, 31, 32, 39, 44)and wet oxidation ( 5 , 6, 16, 17, 35). Alkaline fusion in Pyrex test tubes is suitable, provided the fusion temperature is not alloFed to rise above
:\ -
350
Y
I I
200
Figure 2.
WOO
1300 1200 1100 I000
900 8 00
700 600
SO LV E N T S
0
Y
t:500
8
zt;400 E
c
W X
300
200
IO0
300
400 WAVE LENGTH IN MILLIMICRONS
A
- CH
L 0R 0F 0 R M
X - E T H A N O L (95%)
1500
200
-WATER
0 -
-A
.
sw
s_
I
S 00
Figure 1. Absorption Spectra of Iodine Dissolved in Toluene, Benzene, and 5 % Aqueous Potassium Iodide
300
400 500 WAVE LENGTH I N MlLLlMlCROUS
600
Absorption Spectra of Iodine Dissolved in Water, Chloroform, and 95% Ethanol
450" to 475" C. Thus, a large number of test tubes may be placed in the oven simultaneously. Etched tubes must not be used. The final problem of transferring the iodine to a potassium iodide solution remained. The most commonly used method for iodine transfer, distillation (30, 87, 33, 53), was discarded because in one apparatus only one distillation may be carried out at one time, the procedure is tedious, and the final volume in which the iodine is contained is too large for the present purpose. The authors finally resorted to extraction procedures, using those solvents wherein the partition coefficient favored complete extraction. The iodine was therefore converted to iodate by alkaline permanganate and then liberated by the action of iodate ion on iodide ion in acid solution, extracted with chloroform, and re-extracted from the chloroform with 5% potassium iodide solution. The potassium iodide solution can then be read in the Beckman spectrophotometer a t 352 or 289 mLc with the I-ml. quartz cuvettes or in a Coleman spectrophotometer using the 3-ml. cuvettes with the 5-cm. light path a t 352 mp. As Shown in Figure 3, the light absorption of iodine in potassium iodide solution a t 352 mp follows Beer's law. This figure also shows that after iodine has been extracted with chloroform and then re-extracted with potassium iodide solution, the curve obtained is also a straight line, This indicates, as would be expected, that the partition coefficient has an effect which is constant for different concentrations of iodine. The
V O L U M E 21, NO. 8, A U G U S T 1 9 4 9
lo07
.zoo.
>
t
u) w 2
a
.IO0
b--b
- IODINE
IN 5%
KI AT 352my
+ - IODINE EXTRACTED BY Soh KI AT 352mY
1
I
0
1
I:o 210 CONCENTRATION I N MICROCRAMS PER MILLILITER
3!0
Table I. Change in Extinction Coefficient for Iodine in Potassium Iodide Solution with Change in Potassium Iodide Concentration (I 1% K I
I. mu.
352
Extinction Density coefficient 0.213
1000
- 2.13 y
per
ml.)
10% K I
5 % KI
Extinction Density coefficient 0.217
1018
Extinction Density coefficient 0.226 1060
differelice in the slope of the two lines is ail over-all measure of the partition coefficients between the solvents. By this method 0.1 microgram of iodine per ml. can be detected and 0.8 microgram per ml. can be determined. To obtain the amount of iodine in the original sample these values should be divided by the factor of 6, inasmuch as the iodine in the original sample is oxidized to iodate which in turn liberates six equivalents of iodine ( 2 , ‘7) to be read on the spectrophotometer. Thus approximately 0.02 microgram of iodine in the original sample may be detected and approximately 0.2 microgram determined. In order to simplify this procedure, the use of toluerie solutions of iodine was investigated at the absorption maximum, for this would eliminate an extra extraction and it ivould not be necessary to prepare potassium iodide solutions daily. Toluene also has the advantage of a lower density than water. Thus, an aliquot may he removed from the upper layer without fear of contamiiiating the pipet by the aqueous layer. Iodine is more soluble in benzeiie anti tulueue than ill chloroform. Moreover, c.hlorofoi,m does not shox a peak worthy of use for iodine determination foi. the entire range of the Beckman spactl.oI-’hotonieter. Iodine in aromatic hydrocarbon solvents shows relatively high peaks in the near ultraviolet (see Figure 1 I. This may iritiicate compound formation. Figure 4 shows two curves. The cui obtained by plotting iodine concentration in toluene against density at 311 mp follo\vs a straight line as far as it was investigated (to 10 micrograms per nil.). The second curve, the standard curve for analysis, is obtained by liberating iodine by the action of known amounts of potassium iodate on excess acidified potassium iodide solution followed by extraction with toluene. This curve is also a straight line, indicating that the partition coefficient between the aqueom and toluene phases is constant for different amounts of iodine. From Figure 4 it is apparent that 1.0 microgram of iodine can be determined. This method is therefore adequate for the determination of 0.2 microgram of iodine, if it is first converted to iodate by permanganate. Benzene was similarly studied arid found to yield a straight line when concentration was plotted against) density a t 300 mp with the hydrogen lamp and 305 mp with the tungsten lamp. Toluene was chosen as‘ the preferred solvent because of its higher hoiling point. To test this procedure, determination$ were carried out on solutions of potassium iodide. The iodide ion was oxidized to iodate with alkaline permanganate. Excess permanganate was destroyed by hydrogen peroxide, and the manganese dioxide formed was subsequently removed by centrifuging. aliquot was taken from the supernatam liquid. Potassium iodide and acid were added to this aliquot to liberate the iodine. The iodine was extracted with a measured volume of toluene. Thr toluene extract was then read directly a t 311 mp against a toluene extract of distilled water which had been carried through the oxidation steps as for thr unknown.
* - IODINE -
M
CONCENTRATION
Figure 4.
IN TOLUENE AT 311 m p
IODIUE EXTRACTED BY TDLUENE AT 311 m p
IN M I C R O G R A M S PER MILLILITER
Density of Toluene Solutions of Iodine Before and after extraction procedure
Table I1 shows the amourit of iodine recovered from knon-n concentrations oi potassium iodide on 28 analyses rut] consecutively. The results are indicative of the accuracy that may be obtained under routine conditions. It is apparent from this series of determinations and numerous others, thai when 0.2 microgram of iodine is being
1008
ANALYTICAL CHEMISTRY
Table 11. Iodine Recovered from Potassium Iodide Solutions Sample No. 1
2 3
? 6 7
8 9 10 11 12 13 14
Iodine Iodine Added, Recovy ered, y 0.79 0.79 0.79 0.79 0.40 0.40 0.40 0.40 0.29 0.29 0.29 0.29 0.20 0.20
0.79 0.78 0.78 0.77 0.40 0.40 0.38 0.37 0.29 0.29 0.29 0.27 0.20 0.20
Iodine Re- Sample Added. covered No. y 100.0 98.7 98.7 97.5 100.0 100.0 95.0 92.5 100.0 100.0 100.0 93.1 100.0 100.0
15 16 17 18 19 20 21 22 23 24 25 26 27 28
Mean Yoreeovery = 98.3. Average deviation from mean % recovery
0.20 0.20 0.15 0.15 0.15 0.15 0.10 0.10 0.10 0.10 0,080 0,080 0.080 0,080
Iodine Recovered. y
% Recovered
0.19 0.19 0.15 0.15 0.15 0.16 0.091 0.091 0.091 0.11 0,080 0.080 0.066 0,091
95.0 95.0 100.0 100.0 100.0 106.7 91.0 91.0 91.0 110.0 100.0 100.0 82.5 113.8
= 4.2.
determined, accuracy within 5y0 niay be obtained regularly with the toluene extraction procedure. The average deviation from the mean per cent recovered includes determinations on less than 0.1 microgram, which is apparently beyond the range of this method. However, these results were included to indicate the fact that such amounts of iodine can be detected. Because of the varying results that are obtained in determining protein-bound iodine in serum, depending upon the method employed in precipitating and washing the serum proteins, these results are not included in this study. The protein-bound hormone iodine in serum is being investigated and will be reported in a separate study. REAGENTS
Potassium Iodide Stock Standard. Anhydrous potassium iodide (analytical reagent, 1.308 grams) is made up to 1 liter. 1 ml. == 1 mg. of iodine. One milliliter of this solution diluted to 100 ml. is equivalent to 10 micrograms per ml. Potassium Permanganate, 1%. One gram of the analytical reagent is made up to 100 ml. with distilled -rater. Sodium Hydroxide, 1%. One gram of the analytical reagent is made up to 100 ml. with distilled water. Hydrogen Peroxide, 6%. Hydrogen peroxide (3Oy0,reagent grade) is diluted to five times its volume yith distilled water. Sulfuric Acid, 5940, is prepared by diluting 5 g r a m of analytical grade concentrated sulfuric acid to 100 ml. with distilled water. Toluene, analytical reagent. Potassium Iodide, 1%. One gram of potassium iodide (analytical reagent) is made up to 100 ml. Kith distilled water. Tliis solution is best made up fresh daily. Potassium Iodide, 5%. Five grams of the analytical reagent are made up to 100 ml. This solution is best made up fresh daily. PROCEDURE
The potassium-iodide solutions to be determined (0.2 ml. .each) are pipetted into 5-ml. centrifuge tubes with a mark a t 2 ml., and 0.1 ml. of the 1% sodium hydroxide solution is added, followed by 0.1 ml. of 1% potassium permanganate. If protein is present, it is dissolved in 1% sodium hydroxide in Pyrex test tubes, brought to dryness in a 100” C.oven, and ashed at 450’ to 475’ C. The residue is redissolved in water and a 0.2-ml. aliquot is treated as for the potassium iodide solution. The contents of the tubes are mixed, and the rack with the tubes is placed in a boiling water bath for 30 minutes. The rack is removed from the boiling water bath, allo-red to cool t o room temperature, and placed in a refrigerator until the tubes have reached refrigerator temperature. Cold 670 hydrogen peroxide is added from a dropper to each tube, which is kept immersed in a beaker of cracked ice and salt during the decolorization of the permanganate. If these precautions are not followed, excessive amounts of peroxide will be needed, for the precipitated manganese dioxide catalyzes the decomposition of hydrogen peroxide a t room temperaturcs The tubes are now placed in a 37” C. oven for 1 hour, or allowed to stand a t room temperature overnight to decomposc excess peroxide. The volume is made up to the 2-ml. mark, and the tube is shaken and then centrifuged at 2600 r.p.ni. for 15 minutes. A 1.5-ml. .aliquot is taken, preferably by filteririg thc supernatant liquid
into another test tube bzfore taking the aliquot, so as to ensure complete separation from the manganese dioxide. Method A. The 1.6-ml. aliquot is transferred to a 12-ml. centrifuge tube with ground-glass stopper. Silicone grease may be used to prevent leakage when shaken. Then 0.1 ml. of 1% potassium iodide solution is added, followed by 0.2 ml. of 6qo sulphuric acid; 1.8 ml. of toluene are added, and the tubes are shaken in a mechanical shaker for 10 minutes. The tubes are now centrifuged at 2000 r.p.m. for 5 minutes. The toluene layer is transferred to 1-ml. quartz cuvettes and then read on the Beckman spectrophotometer a t 311 mp. Method B. Tile 1.5-nil. aliquot is transferred to a centrifuge tube with ground-glass stopper, and 0.1 ml. of 1% potassium iodide solution is added, followed by 0.2 ml. of 5% sulfuric acid. Then 1.8 ml. of chloroform are added, and the tube is shaken for 10 minutes in a mechanical shaker. The tube is centrifuged a t 1500 r.p.m. for 3 minutes. The upper layer is aspirated off, 1.5 ml. of the lower layer are transferred to a second centrifuge tube with ground-glass stopper, 1.5 ml. of 5y0 potassium iodide solution are added, and the tube is shaken for 10 minutes. The tube is centrifuged as before, and the upper layer, potassium iodide solution, is transferred to 1-nil. cuvettes and then read on the Beckman spectrophotometer a t 352 mp. For the Coleman spectrophotometer, 3.5 ml. of 6yo potassium iodide solution are used for the extraction. The reading may then be made with the 3-ml. cuvettes with a 5-cm. light path a t 352 nip. LITERATURE CITED
Aldridge, W.N., Analyst, 70, 474 (1945). -4ndrew.s. L. W.. J . Am. Chem. SOC.,25,756 (1903). Bland and Lloyd, J . SOC.Chem. I n d . , 32,948 (1914). Brode, I\’. R., J . Am. Chem. Soc., 48, 1877 (1926). (5) Christensen, B. G., Ugeslzrift Laeger, 105, 866-9 (1943). (6) Cook, J . Chem. Soc., 47,471 (1885). (7) Dietz, H . , and Margoshes, B. M., Chem. Ztg.. 28, 1191 (1904). (8) Dupasquier, .4.,Ann. chim. phys., 73, 310 (1840). (9) Englis, D. T., and Knoepfelmacher, 9..4.,IND.ENG.CHEM., ANAL.ED.,17, 393 (1945). (10) Fajans, K.. and Hassel, O., 2. Eleiztrochem., 29, 495 (1923). (11) Fajans, K., and Wolff, H., Z. anorg. allgem. Chem., 137, 221, 233 (1924). (12) Fellenherg. T. von. Biochem. Z., 152, 116 (1924). (13) Flox, J., Pitesky, I., and Alving, A. S., J . Bid. Chem., 142, 147-57 (1942). (14) Geelmuyden, Z . anal. Chem., 35, 503 (1896). (15) Grauer, R. C., and Saier, E., Endocrinology, 24, 553 (1939). (16) Groak, B., Biochem. Z., 175, 455 (1926). (17) Ibid., 270, 291 (1934). (18) H a h n , F. L., and Adler, M.,Proc. 8th Am. Sci. C G ~ Q7, . , Phys. and Chem. Sei., 169-75 (1942). (19) Hilty, W.W., and Wilson, D. T., IND.ENG.CHEY.,A x . 4 ~ .ED., 11, 637 (1939). (20) Jannasch, Ber., 39, 196 (1906); J . pralzt. Chrm., 78, 28 (1908). (21) Kohler, B., Chem. Listu, 14, 137-40, 195-9 (1920). (22) Kolthoff, I. &PI h. a r, m . Weekblad., 54, (1917); 58,917 (1921). [23) Ibid., 57, 836 (1920). (24) Kolthoff. I. M., 2 . A n a l . Chem., 60, 451 (1921j. (25) Kolthoff, I. M.,Jlenzel, H., and Furman, N. H., “Volumetric Analysis,” Vol. 11, .~ pp. 348-52, New York, John Wiley & Sons; 1929. (26) Kolthoff, I. hl., and Sandell, E. B., “Textbook of Quantitative Inorganic Analysis,” pp. 618-21, New York, Maemillan Co., 1943. (27) Lelihvre, J., and Jlenager, Y.,Compt. rend., 178, 1315-16 (1924). (28) Liebig, Liebig’s Ann. Chem., 185, 289, 307 (1853). (29) Lottermoser, A,, Seiferr, W.,and Forstmann, W., Kolloid Z., Zsigmondy Festschr., 36, 230 (1925). (30) RlcClendon, J. F.. “Iodine and t h e Incidence of Goiter,” C h a p . 111, University of Minnesota Press, 1939. (31) McClendon, J. F., J . Biol. Chem., 102, 91 (1933). (32) McClendon, J. F., and Remington, R . E . , J . Am. Chem. Soc., 51, 394 (1929). (33) RZcHargue, J. 8.. Young, D. W., Roy, W. R., ISD.ESG.CHEY.. ANAL.ED.. 4, 214 (1932). (34) Martindale, W. H.. “ E x t r a Pharmacopoeia,” Vol. 11, p. 86, London. H. K . Len-is & Co., 1929. (35) Rlatthews, X . L., Curtis, G. M., and Brode, TI7. R., ISD.ENG. CHEY.,As.%L.ED.,10,612 (1938). (36) Rlessinger, Bpi-., 21, 3366 (1888). (37) Mohv, Fv., “Lehrbuch der chemische analytische Titrierenmethoden,” Braunschwerg, S.Vieweg & Sohn, 5th ed., Vol. 2, p. 81, 1859. (38) Mullet, and Diffenthsler, Z . anorg. allgem. Chem., 67, 418 (1910). (1) (2) (3) (4)
V O L U M E 21, NO. 8, A U G U S T 1 9 4 9
1009
(39) Perkin, H. J., Brown, B. R., and Lang, J., Can. M e d . Assoc. J . , 31, 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); 39, 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, 159-70 (1942). 147) Sheiill, M. S.,Z . physak. Chem., 47, 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 (1907). (50) Taurog. A., and Chaikoff, I. L., J . Bid. Chem., 163, 313-22 (1946). (51) 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). RECEIVED June 9, 1948. Part of a dissertation presented by John J. Custer to the Graduate Faculty of Brooklyn College in partla1 fulfillment of the requirements for the 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.
