1199
V O L U M E 20, NO. 12, D E C E M B E R 1 9 4 8 SUMMARY AND CONCLUSIONS
Table VI.
Contact Time, Min. 15 30 60 120
1
15
30 60
120
Effect of Acidification on Residual Chlorine in Sewage ( p H 7, room temperature) Residual Chlorine, P.P.M. Acid o-Tolidine titration R a w sewage, chlorine dose 12 p.p.m 0.10 0.68 0.02 0.34 0.00 0.00
0.00 0.00
Sewage effluent, chlorine dose 6 . 5 p.p.m 0.75 3.56 0.60 2.07 0.40
0.18 0.05
Piorm titration 4.75 3.87 2.69 0.83
5.46 4.11
0.68
2.56
0.00
1,86 0 85
0.35
typical samples of raw sei5age and of effluent will serve as an illustration. Samples of each were chlorinated in the laboratory. At various contact times aliquots were removed, each of which was analyzed for residual chlorine by the three methods. The results are given in Table VI. With this particular sample of chlorinated raw sewage, dissipation of available chlorine on acidification causes the error in the o-tolidine test. Previous acidification causes the titration to give practically as lon a result as the o-tolidine method. The titration of the effluent sample after acidification is significantly lower than the normal titration but higher than the o-tolidine result. Both sources of error are about equally important for this particular effluent sample.
On solutions of pure amino compounds low results in the o-tolidine method for residual chlorine can be caused either by loss of available chlorine on acidification or by slow rate of production of color. Both mechanisms may contribute to the errpr in the method as applied to sewage. When a sample of chlorinated sewage or amino compound containing a certain amount of residual chlorine according to amperometric titration is partially dechlorinated, the resulting rcsidual chlorine concentration found by amperometric titration agrees very well with that calculated from the quantity of dechlor used. Even though the residual chlorine indicated by the otolidine method on the same sample is a small fraction of the total and also of the amount removed by dechlorination, the mading with o-tolidine does not become zero. It is concluded that the residual chlorine measured by the o-tolidine test is not ncwxsarily the most reactive portion. Because there is more than one independent cause for the error in o-tolidine reading, this error will not be a constant one. LITERATURE CITED (1) rim. Public Health Assoc., New York, "Standard Methods for Examination of Water and Sewage," 9th ed., 1946. ( 2 ) Elder, A. L., and Buswell, -1.M.,Ind. Eng. Chem., 21, 560-2 (1929). (3) Lea, C., J . Soc. Chem. Ind., 52, 245-50T (1933). (4) Marks, H. C., Joiner, R. R., and Strandskov, F. B., Water R. Sewage W o r k s , 95, 175-8 (1948). (5) Palin, A. T., Analyst, 70, 203-7 (1945). RECEIVED l l a y 3, 1948. Presented before t h e Division of Water, Sewage. CHEMICAL a n d Sanitation Chemistry a t the 113th l l e e t i n g of the A M E R I C A N SOCIETY, Chicago, Ill.
Polarographic Determination of Folic Acid W. J . MADER
AMI
H. 1. FREDIANI, Merck & Co., Znc., R a h t c q , X . J .
Folic acid may be determined quantitatively and rapidly by polarographic means. At a pH of 9 to 9.Sy in 1% tetramethyl ammonium hydroxide E '/2 us. S.C.E. = 0.98 volt and the diffusion current constant = 1.72. The use of cadmium as an internal standard permits a precision of *29& irrespective of ordinary temperature (=!=lo"C.) and drop rate (0.75 second) changes. The method to folic acid tablets and tablets with Bg added. I t cannot he may be applied __ used in the presence of iron.
D
ESPITE numerous publications within recent years and widespread interest in folic acid (pteroylglutamic acid, vitamin Bc), a rapid method for the determination of this vitamin has been much desired. In this laboratory a polarographic method has proved sufficiently simple, precise, and specific for use on relatively pure folic acid as well as known mixtures of folic acid and the necessary constituents for the formation of various tablets. Although the authors have not been in position to compare their method with the only other nonmicrobial assay JO far suggested for this liver L. c a m factor ( I ) , it is offered as a viorkable, rapid assav method for chemists interested in folic acid. PRINCIPLES OF METHOD
The pure folic acid, or preparation containing it, is dissolved in tetramethyl ammonium hydroxide solution containing cadmium chloride as internal standard. Sufficient ammonium chloride is used to prevent precipitation of the cadmium from the alkaline solution. A polarogram of this solution yields two clearly defined waves, one a t 0.74 volt (against the saturated calomel electrode) for the cadmium and one a t 0.98 for the folic acid (Figure 1). With known folic acid concentrations (and fixed cadmium concentration) a straight line results in plotting the step-height ratios of cadmium-folic acid against folic acid concentration using log log coordinates (Figure 2).
RE4GENTS
Place 100 ml. of lOy0tetramethyl annnonium hydroxide (Eastman KO.1515) in a 1-liter volumetric flask, and add 11 grams of reagent ammonium chloride and 500 ml. of distilled water. When solution is complete add 115.0 grams of reagent cadmium chloride (CdC12 2.5 H20, carefully weighed) and shake until the solutio11 is clear. Add 10 ml. of 0.1% alcoholic methyl red as maximum suppressant and dilute t o the mark with distilled water. The concentration of ammonium chlorids used (approximately 0.2 X )is not critical; a 0.1 -11' solution provided sufficient buffering action to prevent precipitation of the cadmium. The concentration of tetramethyl ammonium hydroxide is also not a critical faotor, as the curves obtained with a standard folic acid sample were not affected by a 507, change in concentration (increase or decrease) of this constituent. It is, of course, necessary that the solution be definitely alkaline. The final concentration of methyl red used (0.001%) has been found in this laboratory sufficiently strong to eliminate maxima effect. Decrease of this concentration to 0.0005% permitted occasional maxima to be observed in the cadmium wave. The weight of cadmium chloride used was such as to provide a cadmium wave height comparable to that of the folic acid. I n this way physical measurement er-
1200
ANALYTICAL CHEMISTRY
rors (wave height measurements on the polarograms) are of the same order of magnitude. For samples very low in folic acid concentration a 1 to 10 dilution with respect t o cadmium ion was found necessary, so t h a t higher instrument sensitivities could be used and the wave height due to the cadmium 11 ould not be disproportionately large compared to t h a t due to folic acid. Careful weighing of reagent grade cadmium chloride provides sufficient control of the cadmium concentration. This is essential, when fresh lots of solvent are prepared, t o obviate the necessity of recalibrating each lot of solvent prepared. This solution is used as solvent for the folic acid samples.
Table I.
Reproducibility of Replicate Samples
Folic .Icid Concn.. ~lg./Ml. 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30
Ratio of C d to Folic Acid 1.32 1.40 1.31 1.28 1.39 1.36 1.35 1.38 1.36 1.33
Table IT.
Drop Rate, Sec./Drop 3.33 4.62 2.72 3.52 3.52 3.52 3.52 3.52 3 52 3 , .52
Temperature 0 C. 27 28 28 4 28 28 28 28 18 28
Analysis of Folic Acid Tablets
(Each tablet prepared to contain 10 mg. of folic acid as determined by microbial assay) Folic Acid Found, IIg. per Tablet Type of Tablet 8.7 Folic acid 8.7 10.5 Folic acid and synthetic vitxniin H 10.5 complex '3 7 Folic acid and iron 18
__
and folic acid concentration of 1.0 nig. per ml. l t this sensitivity 1-mm. deflection of the recorder corresponded t,o 0.08 microampere. DISCUSSION
.ten1 herein described is singularly lacking i n vhanges of tjoth temperature and mercury drop rate. The p H of the finid solution&as measured with a high p H glass electrode was approximately 9.3. The findings of Stokstad et al. ( 7 ) that folic acid in alkaline solution is relatively stable to light were confirmed. Polarograms reproducible to 2y0were obtained o n a solution that was permitted to age for 24 hours on the 1ahoi.atory shelf b e t m e n determinations. Replicate runs on a fixed sample (Table I) indicate a reproducihilit,y of * 2 c & if the drop rateis controlled within 0.75 second and temperature within 10" C. Mercury drop rate and temperature were purposely varied as indicated. These variations exceed the conditions ordinarily encountered in an analytical laboratory. The diffusion current constant as defined by Lingane ( 4 ) was calculated to be 1.72. It rvas found more convenient to utilize the ratio-conrentration curve and thus to read off sample concentrations directly in milligrams por milliliter than to calculate sample concentrations. The folic acid sample ujed for calibrating purposes \vas believed, lly other criteria, to be of high purity. Elementary analysis gave the following results:
Figure 1
Sample 51.48 4.54 22.68
C H S
I
05
I
1.0 V O L T S Figure 2
I
15
A sufficientlylarge sample should be taken so t h a t the final solution will contain approximately 1.0 mg. of folic acid per ml. It has been found convenient in this laboratory to use 25-mg. samples of relatively pure material (weighing into 100-ml. beakers) and to pipet into the beaker exactly 25 ml. of the solution. For tablets prepared t o contain 10 mg. of folic acid per tablet 5 tablets were added to 50 ml. of solvent and agitated until disintegration was complete. It was not necessary to filter off starch or other tableting material, as long as complete dissolution of the folic acid was effected. Oxygen was removed before polarizing by bubbling nitrogen through the solutions for 5 minutes. The polarogram shown in Figure 1 was taken on a Sargent Model XX polarograph with a sensitivity setting of 2-10, initial voltage of 0.0, span voltage of 2.0, damping switch at position 2,
Theory 51.69 4.33 22.21
The ultraviolet absorption of this sample (0.0343 gram dissolved in 25 ml. of 0.1 S sodium hydroxide and diluted to 250 nil. in ethanol) indicated: AIM
260 287 367 (plateau)
cm. 157, 24,200 27,000 8,200
Three types of tablets were available to the authors for study: tablets prepared to contain approximately 10 mg. of folic acid as sole active ingredient, tablets prepared t o contain both folic acid and B complex as active ingredients, arid tablets prepared to contain folic arid and ferrous iron as active ingredients. The results obtained on typical routine samples of these tablets are indicated in Table 11. The high results obtained with the tablets containing iron iiidicated that ferrous iron interfered Ti-ith this method. This mas found to be true by adding 1 mg. of ferrous sulfate per ml. t'o a st,aridard folic acid solution. The polarographic result indicated
V O L U M E 20, NO. 12, D E C E M B E R 1 9 4 8 2.35 nig. of folic acid instead of the 1.0 taken. This interference is slio~vnas a direct enhancement of the folic acid wave and is caused by the direct superimposition of the iron wave onto that of folic. acid. .4s found in this laboratory, the half-wive potential of thc ferric-ferrous reduction v a s -0.94 volt with the tetramethyl aninn~niumhj-tlroxide solution used. This was to be expected, since half-Ivave potent,ial for ferric-ferrous iron in potassiuni hydroside and mannitol has bcrn reported as -0.9 volt (J). Although it is possible that use of au oxalate medium ( 8 ) may shift the iron half-wave so that i t ~vouldno longer interfere. this of the problem was of minor importance in this laboratory and sufficient t i n e could not br devoted to its solution. Ttilizirig higher instrument sensitivit,y with correspondingly lon-er radmium ion concentratioris, it was found feasible to test for COIIrenti,atiorir oi’ folic acid a‘ low as 1 mg. per liter. This sensitivity is not LO great as with the turbidimetric microbial method of Rohcrts and Snell (6, 10 micrograms per liter). The latter nitathod, however, required a minimum of 16 hours per aqzaj- as ronip:wed to 10 to 15 minutes for the polarographic, 1~roc~diire. In thr. rcrcwt study of drgradation prodncts of rhiztq)tmin by
1201
Rickes et al. (6) polarographic studies indicated a general pterin class reduction at approximately 0.8 volt in 0.1 JP lithium borate ( p l l 9.12) buffer. Thus, in a study of folic acid of presumably high purity pterin contaminants should be considered as likelv to intei fere with polarographic assays. LITERATURE CITED
Hutchings, B. L., Stokstad, E. L. R., Boothe, J. H., Momat, J. H., Waller, C. W., Angier, R. B., Semh, J., and Subbarow, J., J . B i d Chem., 168, 705-10 (1947). Kolthoff, I. M., and Lingane, J. J., “Polarography,” p. 170, New York, Interscience Publishers, 1941. Ibid., p. 484. Lingane, J. J., IND.ENG.CHEM.,ANAL.ED.,15, 553 (1943). Rickes. E. L..Trenner, N. R., Conn, J. B.. and Keresztesy. J. C., J . Am. Chem. SOC.,69,2751 (1947). lioberts, E. C., and Snell, E. E., J . B i d . Chem., 163, 499 (1946). Stokstad, E. L. R., Fordham, D., and de Grunigen, rl., I b i d . , 167, 877 (1947).
RECEI\-EI) X a y 11, 1948. Presented before the Division of Analytical and CHEMICAL S O C I E T Y . l\Iicro Cheiiiiatry a t the 113th hleeting of the AXERICAX Chicago, Ill.
CHEMISTRY OF THORIUM Quantitative Estimation of Thorium by Precipitation with Radioactive Pyrophosphate ‘I’lIEKILI) TIOELLEK
.&%Rin
GEORGE I
