ANALYTICAL CHEMISTRY
1408 ference with the phenolphthalein color change. In fact, interferences are likely to occur in the presence of any anion derived from a very weak acid, owing to its strong buffering action. Bromide, iodide, cyanide, and thiocyanate may be removed, prior to analysis, with silver nitrate, and excess silver precipitated with sodium chloride. Thiosulfate may be oxidized to sulfate before determination of mercury. None of these procedures has been tested so far. Phosphates also interfere with the estimation of mercury by the present method. Phenolphthalein gives no sharp color change, and a mixture of phenolphthalein (3 parts) and a-naphtholphthalein (1 part), though somewhat better, is not entirely satisfactory, Hence phosphate should be removed before analysis, and removal can be very conveniently effected as follows. To the solution containing mercury and phosphate, acetone and then excess alkali are added. Mercury oxide remains in solution and if other heavy metals are absent, phosphate will remain in solution as alkali phosphate. Barium nitrate is then added till the phosphate is completely precipitated as barium phosphate. Sodium carbonate solution is now added to precipitate the excess barium. The filtrate is acidified with nitric acid and boiled to remove carbon dioxide, and mercury is estimated by the general procedure. The method has been tested in the presence of sodium phosphate and found to give excellent results (Table 111). Though barium nitrate has been used by the author, other suitable alkaline earth or heavy metal salts should serve the purpose equally well. If an alkaline earth or heavy metal salt is already present in the solution, the phosphate will be partly or completely precipitated out on addition of alkali. If necessary, complete removal of phosphate may then be effected with barium nitrate as above. This affords a very convenient and rapid method for separation of mercury from phosphate. As this work was in progress, the author’s attention was drawn to a paper by Fernandez, Snider, and Rietz (6),who have used
acetone-water mixture for analysis of mercuric ion-anion mixtures. They have estimated mercury by direct titration with alkali in aqueous acetone using phenolphthalein as indicator, in the presence of added chloride ion. In case the solution contains free acid, they first neutralize it to the bromocresol green end point and then titrate the mercury with phenolphthalein as indicator. They have obtained very good results. The present author has, however, experienced difficulties in detecting the correct end point under such conditions, as the color change of phenolphthalein is not very sharp, and is further masked by the presence of bromocresol green. The method described in the present paper has a wider field of application for determination of mercuric salts in the presence of free acids, as well as salts of other heavy metals and alkaline earth metals, and phosphates. ACKNOWLEDGMENT
The author is indebted to Santi R. Palit for his kind interest in the work. LITERATURE CITED
(1) Andrews, L. UT., Am. Chem. J . , 30, 187 (1903).
(2) Biilmann, E., and Thaulow, K., Bull. soc. chim.,29, 587 (1921). (3) Britton, H. T. S., Ann. Repts. Progr. Chem., 40, 45 (1943). (4) Britton, H. T. S., J . Chem. S o c , 127, 2110, 2120, 2142, 2148 (1925). (5) Fernandez, J. B., Snider, L. T., and Rietz, E. G., -4N.4~. CHEM., 23,899 (1951). (6) Gilchrist, R., J . Research Nail. Bur. Standards, 30, 89 (1943). 17) Kolthoff. I. -11.. - , and Keiizer. J.. Pharm. Weekblad. 57.911 (1920). (8) Rupp, E., Chem. Ztg., 32,1077 i1908). (9) Rupp, E., and Lehmann, F., Pharm. Ztg., 52, 1014 (1907). (10) Rupp. E., Muller. K., and Maiss, P., Ibid., 67, 529 (1926). (11) Skr-amovsky, S., and Uzel. R., &sopis 8eskosZov. Ldkdrnictva, 14,33 (1934). (12) Whitmore, F. C., “Organic Compounds of Mercury,” p. 157, New York, Chemiral Catalog Co., 1921. lean X 3.54 Standkrd deviation, o 0.031 Coefficient of variation, V , % +o. 9
+o. 8
0,800
0.600
Recovery.
0,0189 0.0189 0.0472 0.0944 0.472
% 0.0184 0.0184 0.0490 0.0995 0.475
0.0189 0.0189 0.0474 0.0944 0.472
0.0186 0.0193 0.0463 0.0950 0.463
98.4 102.1 98.1 100.6 98.1
0.0189 0.0189 0.0472 0.0944 0.472
0.0196 0,0192 0.0498 0,0990 0.480
103.7 101.6 106.5 104.9 101.7
D.B.
0.0189 0,0189 0.0472 0.0944 0.472
0,0188 0.0196 0.0495 0.0965 0.468
99.5 103.7 104.9 102.2 92.2
H.H.
0 0189 0 0189 0 0472 o ow4 0 4i.2
0.0184 0.0191 0.0475 0.0944 0.478
97.4 101.1 100.6 100.0 101,3
0.0189 0.0189 0.0472 0.0944 0.472
0.0184 0.0188 0.0483 0.0945 0.470
97.4 99.5 102.3 100.1 99.6
0.120 0.60 0.60
0.047 0.049 0.162 0.141 0.154 0.158 0.272 1.41 1.38
92.2 96.1 111.0 96.6 99.4 100.0 98.6 101.4 99.3
0.036 0.060 0.120 0.360
0.0344 0.058 0.115 0.352
95.6 96.7 95.8 97.8
Code D.F.
%
J.B.
M.C.
The calibration curve ha. :L slope of approximately 0.02 (extinction unit) for each microgram per milliliter of polyvinylpyrrolidone and an intercept approximating the extinction value of the iodine reagent. This value is about 0.2 (in the dilution used in the procedure) and if the reagent is stored stoppered and in the dark, it remains constant for several weeks.
Table I.
Total PVP Found,
PVP Found,
Clinical samples of urine of human patients receiving P V P infusions
Normal dog urine
...
PVP Added,
%
73
0.027
0.024 0.024
71
0.086
79 83 85 81
0.095 0,098 0.156 0.790
0.060 0.060 0.060 0.060
1
2 3 4
.
% 97.4 97.4 103.8 105.4 100.6
The color intensity is somewhat dependent on the molecular weight or K value of the polyvinylpyrrolidone preparation, ae shown in Figure 1. For this reason a calibration curve should be used which corresponds to the K value of the sample to be analyzed. In hot weather the iodine reagent may deteriorate during performance of the analysis. Therefore the reagent, sample, and spectrophotometer compartment should be cooled with tap water to a temperature not exceeding 20" C. Some urines have a strong bleaching action on the polyvinylpyrrolidone-iodine complex, which interferes with the analysis. In these cases a higher dilution should be used, despite the decreased precision a t low absorbance. RESULTS AND DISCUSSION
rn
ru
0.400
O.PO0
~~~~
~
~______
~
10 PO PVP CONCENTRATION, MEQ./ML.
30
Figure 1. Calibration Curves for Polyvinylpyrrolidone of Three K Values Arrow indicates value of reagent blank
When the polyvinylpyrrolidone content is determined in saline plasma extender, the inherent precision of the method can be demonstrated (Table I). The results of recovery tests in urine are shown in Table 11. The somewhat larger scatter of results is in part due to the adverse influence of residual bleaching. This bleaching effect, as well as the turbidity of some sera, was essentially overcome by the use of 0.4 M citric acid. It is possible that the body fluids of some test animals may introduce difficulties not encountered in the work reported, in which all samples of human, dog, and rabbit urines and sera yielded to this technique. Results with reconstituted human plasma and sera of various animals, to which known amounts of polyvinylpyrrolidone have been added, are shown in Table 111. The
1410
ANALYTICAL CHEMISTRY
Table 111. Determination of Polyvinylpyrrolidone in Serum PVP Added,
P\’P Found,
Recovery,
0 0155
0 0233 0 W33
0 0152 0 0240 0 0233
98 1 103 0 100.0
Sormal rabbit serum
0 0155 0 0233 0 0233
0 0159 0 0239 0 0235
102 6 102.8 100 9
Reconstituted normal human plasma
0 0155 0 0233
0 0164 0 0230
105.9
%
Type
Xorrnal dog serum
Table IV. Time, Nin. 1
5 15
R
%
98.7
Polyvinylpyrrolidone Concentration in Rabbit Serum (% PVP)
Level 1 1 . 1 9 , 0 . 9 4 , 0.90 1.08, 0 . 8 8 , 0 . 9 1 o 81, 0 . 6 3 , 0 . 7 3
Level 2
Level 3
0.46, 0.32, 0.36 0 . 4 5 , 0 . 3 4 . o 36 0.46, 0.34, 0.38
0.29, 0.34, 0.27 o 76, , . . , 0 . 2 7 0 . 2 5 , . . . , 0.26
0 65, 0.57, 0.35, 0.36, 0.24,
0.32, 0.22, 0.23 0.24, 0.24, 0.24 0 . 1 8 , 0.16, 0 . 1 5 0 13, 0 . 1 2 , 0.11 0.11,0.11, 0 . 0 9
0.19, 0.14, 0.10, 0.08,
Houn 1
4 24 48 72
0.47, 0.51 0 42, 0 . 4 6 0.30, 0.35 0.24. 0.29 0.20, 0.27
0.24, 0 19, O,l5, 0.10, 0.05,0 09,
0.16 0.12 0.08 0.00 0.04
values represent lower physiological ranges and the precision approaches that obtained in water. To illustrate the technique in animal work, results of polyvinylpyrrolidone determination in rabbit serum are included in Table IV. Kine animals received one infusion each of polyvinylpyrrolidone plasma extender. The animals were in three groups of graded dosage levels in the range of usual human dosage, with the highest level at 440 mg. per kg. This experiment was carried out over a 72-hour period, by which time the serum level was in the range of 500 p.p.m. in some animals. The method is suitable for the analysis of body fluids, despite the fact that these are known to take up iodine. The spectral absorption of the polyvinylpyrrolidone-iodine complex is identical with that of the potassium triiodide solution, except for intensity. Horvever, when the complex is formed, some iodine is removed from the solution, so that its own absorption is somen-hat decreased; for this reason no true “blank” is available. The solvent (water) is used instead, and the calibration curve corrects for the reagent blank. While the body fluids do not interfere with the formation of the color complex, they too may consume
some iodine. The method is designed to minimize this effect and to enhance the intensity of the color complex. The small residual reduction of intensity of color of the reagent due to the body fluids appears to be approximately balanced by their color, In unusual instances, where there is an abnormal iodine uptake, faulty results are not likely to be obtained, because this uptake is gradual and the “bleaching” will warn the operator. The main disadvantage of the technique described is the variation of absorbance with the molecular weight of polyvinylpyrrolidone. This defect is common to some degree to the other methods cited. This effect should not be ignored, but its importance is minimized by the fact that commercial polyvinylpyrrolidone plasma extenders have similar characteristics. It is known that the frartions of lower molecular weight (lower K value) are excreted first and the fractions of higher molecular weight are excreted only gradually. For most exacting work this should be taken into account and the polyvinylpyrrolidone content read off the calibration curve corresponding to the K value of the sample. If this is not done, the result may be in error by 10 t o 25%. The sensitivity of the method is high: 5 to 30 p.p.In. of polyvinj~lpyrroliJonein the final dilution, which is adlquate for all practical purposes. Moreover, by using 5-em. cuvettes and the iodine reagent as a blank, this range was mtended down to 1 p.p.m. of pol\~in~lpyrrolidone. ACKNOWLEDGIIEYT
The clinical samplce of human urine were supplied by J. E. Rhoads, University Hospital, Philadelphia, Pa., and the other test samples by J. Y. P. Chen Pharmacology Division, Schenley Laboratories. The authors wish t,o acknowledge their kind cooperation, and to thank Bruno Puetzer and Kurt Ladenburg for encouragement’ and discussions and Srhenky Laboratories, Inc., for permission to puhli~hthip paper. LITERATURE CITED
Chinard, F. P., J . Lab. C‘lin. M e d , 39, 666 (1952). (2) Frijtag Drahhe, C. -1.J. yon. and Reinhold, J., Ibid., 40, 616 (1)
(1952).
(3) Haden, R. L., .l.B i d . Chem., 56, 469 (1923). 24, (4) Levy, G. B., Caldas, I., Jr., and Fergus, D., ANAL.CHEM., 1799 (1952).
( 5 ) Thrower, W.R., and Caniyhell, H., Lamxt, 1951, 1096. (6) Zipf, K., ICZin. Ff’ochachr., 23, 340 (1944). RECEIVED for review Soreinher 14, 1952.
Accepted 118y 8, 1953.
Determination of Acrylonitrile in Colored Solutions G. J. JANZ AND N. E. DLNC4N Department of Chemistry, Rensselaer Polytechnic Institute, Troy, Y. Y . the quantitative determination of acryloliitrile in A -coloredforsolutions arose in the course of studies in progress ~ E E D
in this laboratory concerning the reactions of nitriles and dienes a t high temperatures. The standard analytical procedure for acrylonitrile (1, 2 ) is based on the cyanoethylation of dodecanethiol (dodecyl mercaptan) in the presence of a basic catalyst.
RSH
+ CH-CH-CX
catalyst +
RSCH,-CH?-CN
The excess mercaptan is determined iodometrically, the conipletion of reaction being noted by the appearance of the charncteristic faint color of iodine (yellow). Starch cannot be used to detect this end point, as the characteristic blue complex does not form readily in nonaqueous solutions. To adapt this method
for use with colored solutions in which the above end point is not detectable, a potentiometric titration of the excess mercaptan with silver nitrate was cho-en.
RSH
+ .\g-
-
RS.lg J,
+ H+
For this purpose, the calomel electrode is replaced by a silver electrode in order to determine the end point. The method is thus based on the precipitation of mercaptans with silver nitrate ( 3 ) . The results for precipitation of silver dodecyl sulfide are shown in Figure 1. The curve has the typical steep vertical change and point of inflection a t the end point. The e.m.f. scale in this figure is relative only. Two further changes are necessary in the procedure of the standard method. In place of the bromate-iodide reagent for the iodometric titration, silver nitiate in isopropyl alcohol is re-
