V O L U M E 24, NO. 11, N O V E M B E R 1 9 5 2
1829
the hygroscopicity of lithium chloride and pyridine would have a greater effect in the solutions containing 25% water by volume. The inconsistencies in diffusion constants are not serious if the purity of the organochlorosilane is considered. The purity of the organochlorosilanes ranged from 90 to 99% and the impurities consisted of other organochlorosilanes. This would tend to give either low or high results depending upon the nature of the orgnnochlorosilane present as impurity-Le., whether or not it was a monovhlorosilane or trichlorosilane.
can be accomplished by weighing the organochlorosilane in a sealed glass bulb and breaking the bulb under pyridine in a closed flask. When the substance is completely dissolved in pyridine, transfer to a volumetric flask and dilute to volume with pyridine. From this solution remove an aliquot, such that when it is diluted to 50 ml. i t will fall in the concentration range of 6 X 10-3 &Ifto 0.15 M , and place it in a 50-ml. volumetric flask. Add sufficient pyridine to bring the volume to 25 ml. Add 5 ml. of 3 M potassium chloride and dilute to 50 ml. with water. Place the solution in a polarographic cell and obtain the polarogram by conventional means. Measure the diffusion current and compare the value obtained with a ealibration curve obtained by treating known amounts of hydrochloric acid dissolved in pyridine in the same manner. The calibration curve is a plot of the observed diffusion current against the concentration of chloride ion and is a straight line within the limits of experimental error. The accuracy of this procedure corresponds to an average deviation of 2%. CONCLU SION s
8
0
I6
24
32
40
48
56
7 . Wafer by Volume
Figure 2.
Effect of Pyridine-Water Ratio o n Diffusion Current Constant
Plots of log i / ( i d - 1) against the applied voltage were made for the reduction waves of hydrochloric acid dissolved in 50% by volume pyridine solutions and a straight line with a slope of 0.055 was obtained indicating a reversible reduction involving one electron. Similar plots for the organochlorosilanes also indicated a reversible one electron reduction. PROCEDURE
The reduction of pyridinium ion produced by the hydrolysis of organochlorosilanes in the presence of pyridine can be adapted to the analysis of these substances in terms of their chloride content. Mix a weighed amount of the substance t.0 be determined with dry pyridine in a closed vessel so that the salt formed does not escape as a smoke but dissolves in the excess pyridine. This
Perhaps the greatest limitation of this method is that one organochlorosilane cannot be determined in the presence of others. Only the total chloride content can be determined. If the method is to be used as a purity determination on one organochlorosilane, the presence of other organochlorosilanes or the presence of any substance which hydrolyzes to give an acid will interfere and cause the results to be high. The method can be adapted to the analysis of two organochlorosilanes in the presence of each other, if no other impurities are present and the two organochlorosilanes are knoivn. ACKNOWLEDGMENT
The authors gratefully acknowledge the aid, in the form of a fellowship to one of them (E.A.A.), furnished by the Eastman Kodak Co. LITERATURE CITED
(1) Kolthoff, I. AI., Lee, T. S.,Stocesova, D., and Parry, E. P., A s a ~ CHEM., . 22,521 (1950). (2) Sisler, H. H., Schilling, E. E., and Groves, W. C., J . Am. Chem. Soc., 73,426 (1951). RECEIVED for review March 19. 1952. Accepted August 7, 1952. From a thesis submitted by Earl A. Abrahamson, Jr., in partial fulfillment of t h e requirements for t h e degree of doctor of philosophy In chemistry in the Graduate School of the Cniversity of Kansas, 1951.
Modified Procedure for Determination of Protein-Bound Iodine in Serum HARRY SOBEL AND S . SAPSIN Department of Biochemistry, Division of Laboratories, Cedars of Lebanon Hospital, Los .4ngeles 29, Calif.
protein-bound iodine involves precipitation, digestion, and distillation carried out in separate containers. Recently Conner et al. ( 3 )described an apparatus in which both precipitation and digestion could be carried out. The present report presents a further simplification whereby precipitation, digestion, and distillation are carried out in the same tube, resulting in reduction of the time required for analysis.
Zinc sulfate (ZnSOa.’iH,O), 1.25%, A.C.S. specifications (Baker and ildamson). Potassium iodide, C.P. (Baker). The apparatus (Greiner Glassblowing Laboratory, 3604 East bledford St., Los Bngeles, Calif. j , Figure 1, consists of a digestion tube (22 X 140 mm.) containing a 24/40 standard taper joint and a delivery U-tube (22 X 230 mm. total length and 35-mni. inner distance) containing a standard taper 24/40 male joint a t one end and a 1.25- to 1.75-mm. capillary tube 200 mm. long at the other end.
REAGENTS AND APPARATUS
PROCEDURE
Sulfuric acid, 70% by weight, A.C.S. specifications (Baker and Adamson j. Chromic acid, 60% by weight, 4.C.S. specifications (Merck). Ceric ammonium sulfate, 0.1 X, reagent grade (G. Frederick Smith Co.), in 3.5 N sulfuric acid. Sodium hydroxide, 1% and 0.75 iV, A.C.S. specifications (Merck). Phosphorous acid, 50% by weight, C.F. (Baker and Adamson). .4rsenic trioxide, 0.15 N , C.P. (Baker), in 1.5 N sulfuric acid.
To 0.5 ml. of serum in the digestion tube are added 4.0 ml. of zinc sulfate solution and 0.5 ml. of 0.75 N sodium hydroxide. After thorough mixing the tube is centrifuged for 7 minutes at 1800 r.p.m. and the supernatant is discarded. The contents of the tube are washed with 10 ml. of water and recentrifuged. Only one washing is used in the modified and the Chaney procedures in the authors’ laboratory. The washed precipitate is dissolved in 4.0 ml. of 70% sulfuric acid and 0.5 ml. of chromic acid solution is added. The con-
HE widely used Chaney ( 2 ) procedure for the determination
A N A L Y T I C A LC H E M I S T R Y
1830 tents of the tube are mixed by rotation. Two glass beads are added and the contents of the tube are digested by placing the tube 1.5 to 2.0 cm. above a microburner. The burner is surrounded by a shield of metal tubing 3.0 cm. in internal diameter by 9.0 cm. in height. The flame height is adjusted to 1.5 cm. These conditions must be satisfied to assure the correct digestion period and to prevent bumping. The boiling is vigorous and the level of the boiling liquid may reach to within 2 cm. of the top of the tube. The digestion period takes approximately 5 minutes; the end point is indicated by a sudden decrease in the height of the boiling liquid in the tube. This is usually followed by crystal formation on the walls of the tube and surface of the liquid. This end point is different from that seen in the Chaney procedure. The burner is removed and the contents of the tube are allowed to cool. Two milliliters of water and one glass bead are added. Digestion is once more carried out according to the manner indicated above. Ten minutes after digestion is completed, 4.0 ml. of water and two glass beads are added and the contents of the tube are mixed. One milliliter of 50% phosphorous acid solution is added. The delivery tube is immediately joined with the digestion tube. The capillary tube is immersed in the bottom of a colorimeter tube containing 2.0 ml. of 1% sodium hydroxide.
Table I. Differences between Duplicate Determinations Obtained by Chaney Procedure and by Modified Procedure Differences, y/lOO hI1. 0.0 0.1 t o 0.2 0.2 to 0.4 0.4 to 0.6 0.6 t o 0.8 0.8 t o 1.0 hfean difference, microgram
Duplicate Determinations, % Chaney procedure Modified procedure 26 44 24 22 18 28 6 12 0 16 0
+o.
4
1.5
+0.34
Table 11. Differences between Average Values Obtained by Modified Method as Compared to Chaney Procedure Frequency of Deviation", 3'% Deviations, ? / l o 0 mi. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.8 0.8 1.3 indicates positive deviation; - indicates negative deviation.
+
+
The flame is adjusted to create a uniform boiling in the digestion tube a t such a rate that 55 to 65 bubbles per minute appear in the alkaline solution in the colorimeter tube. The boiling is permitted to continue for exactly 4.5 minutes. A boiling time less than 4 minutes results in low yields, whereas a boiling time over 5 minutes results in the danger of obtaining a green distillate. I t is imperative that during the distillation process every effort be made to maintain uniform heating and to avoid as much as possible all movements of air, such as through open doors and windows, or by movements of personnel in the room other than the analyst. Before the necessity of observing these precautions was recognized, only 70% of the results were satisfactory. The contents of the colorimeter tube are diluted to 10 ml. with water and 0.8 ml. of arsenious acid solution is added. The tubes are immersed in a water bath set at 32' C. After 10 minutes, 0.6 ml. of ceric ammonium sulfate solution is added and the procedure is continued in the usual manner (3). A standard curve is.set u p with 0.02-microgram increments of iodide up to 0.20 microgram.
. i u Figure 1. Apparatus for Determination of Protein-Bound Iodine Table I shows the differences between values of duplicate determinations performed by the Chaney procedure and the modified procedure. The precisions of the two methods Rere compared by the F test ( 5 ) which indicated that the precision of the Chaney method was superior a t the 0.1% level of significance. The confidence limits a t a 95% level for the mean of duplicate analyses ( 4 ) for the modified and Chaney procedures were =k10.9% and zk5.6%, respectively. In Table I1 are shown the differences between the average values obtained by the Chaney procedure and the modified procedure. The new method gave results which, on the average, were 0.072 microgram higher than those obtained by the Chaney method. This difference is not significant as indicated by the t test ( 5 ) . A constant bias of 0.1 microgram would not be of practical significance: The correlation index ( T ) was 0.988. I t is concluded that the modified procedure is satisfactory for the determination of protein-bound iodine in serum and that the normal range of values agrees with those obtained by the Chaney procedure. DISCUSSION
The proposed modification of thr Chaney procedure incorporates the following advantages: Required amount of serum is only 0.5 ml. Quantities of reagents are greatly reduced. Contamination by multiple use of one still is avoided. Running time is halved. The latter point becomes obvious in comparing the time required for the completion of each step in the analysis of 30 individual samples by one analyst.
RESULTS
The results presented in Table I and Table I1 were derived from analytical data obtained from 50 consecutive specimens received in this laboratory. Each analysis was performed in duplicate by the Chaney procedure and the modified procedure. This group included 41 normal values ( 4 to 7 micrograms per 100 ml.). There were 4 values below 4 micrograms per 100 ml. and 5 values above 7 micrograms per 100 ml. These results are in conformity with previous experiences in this laboratory, where approximately 10% of the total determinations yield values which are above or below normal.
Time, Minutes Chanep procedure Xlodified procedure Precipitation and washing Transfer Digestion Distillation
60 0 60 180
Reading Total
375
60
... 30 45
45
180
The results obtained by use of the modified apparatus are considered to be in agreement with those obtained using the method of Chaney.
V O L U M E 24, NO. 1 1 , N O V E M B E R 1 9 5 2 ACKNOWLEDGMENT
The authors wish t o express appreciation t o Richard Henry of Bio-Science Laboratories for his interest in the work and for calculation of the statistical data.
1831 (3) Coimer, A. C., Swenson, R. E.. Park. C. W..Gangloff, E. C.. Leiberman, R., and Curtis, G. &I., Surgery, 25, 510-(1949). (4) Sriedecoi , G. IT.,“Statistical Methods,” Ames, Iowa, Collegiate Press, Inc , 1946. (5) Youden. W. J., “Statistical Methods for Chemists,” New York,
John Wiley &Sons, 1951. REFERENCES
(1) Barker, S.B.. J . Bid. Chem., 173, 715 (1948). (2) Chaney, S . L., ISD. EKG.CHEM.,ANAL.ED., 12, 179 (1940).
R E C E I V Efor D review January 12, 1952. Accepted August 11. 1932.
Presented a t the meeting of the Southern California Section of the American Association of Clinical Chemists, February 5 , 1932.
Separation and Determination of Nicotine and Nornicotine in Tobacco FORREST G. HOUSTOK h-entucky .Igriczcltttral Experiment Station, University of Kentucky, Lexington, K y . I C O T I S E or nornicotine in solution can be determined with a fair degree of accuracy by precipitation as the silicotungstate ( I ) , ultraviolet absorption ( 5 ) ,or titration (3). However, the separation of these two alkaloids provides the greatest difficulty in their determination in i\-icotiana tabacurn. The Bowen-Barthel procedure ( 2 ) is not entirely satisfactory for tobarco samples containing a high proportion of nornicotine, and there is evidence that substances in addition to nicotine and nornicotine which react with silicotungstic acid are obtained by the steam-distillation technique used ( 4 ) . An ext,raction procedure would be expected t o result in fewer decomposition products than steam distillation. The technique for separating nicot,ine and nornicotine described here employs partitioning on a starch column. If a mixture of the two alkaloids is placed on such a column and washed with hexane, the nicotine is readily released and appears in the effluent, Thile nornicotine is completely retained by the column. Sornicotine is then easily removed from the column with chloroform and collected in a separate fraction. .4 titration procedure is described for measuring the alkaloids in each fraction. EXPERIMENTAL
Apparatus. -1column of borosilicate glass tubing 20 111111. by 75 cm. is constricted near one end so as to support a perforated aluminum disk which is covered with a small plug of cotton. Preparation of Column. -In amount of starch equivalent t o 5 grams of moisture-free material is suspended in 35 to 40 ml. of 1butanol sat,urated with water a t room temperature. The starch need not be dried for the preparation of the column. Starch from various manufacturers seems equally suitable. This suspension is poured into the glass column, arid excess solvent is forced out wit,h gentle air pressure until the solvent level is even with the top of the starch column. .1 tight-fitting cotton plug is then forced down the tube until it rests firmly on the surface of the starch. About 25 nil. of h(,rane or Piniilar light hydrocarbon is forced through t h v column in ordrr to n.mh out the excess 1-butanol.
Table I.
Effect of Treatment to Remove .immonia on Nicotine and Nornicotine 5% Nirotine % Sornicotine
5 .ininionis
Added
Treateda rntreated 1.71 1.71 0 10 1.73 1 74 0.00 1.74 1 73 Over concentrated sulfurir acid for 24 hours. 1 00
a
Table 11.
Treated“ 0 22 0.23 0 22
Untreated 0.58 0 25 0 23
Separation of Nicotine and h-ornicotine Added to Alfalfa
Alkaloid Added, 31s. Nicotine Sornicotine
Alkaloid Recox.ered, M g . Sicotine Xornicotine
Preparation of Sample. One gram of the dried and finely ground tobacco sample is mixed with about 0.25 gram of powdered barium hydroxide octahydrate. The mixture is then moistened with 1 ml. of a saturated solution of barium hydroxide and thoroughly stirred. If the tobacco sample contains more than 0.lc; ammonia, it should be left in a desiccator over concentrated sulfuric acid at this point for 15 to 24 hours. This treatment effectively lowers the concentration of ammonia so that it will not subsequently appear in the nornicotine fraction, and there is no detectahle loss of nicotine or nornicotine, as is shon-n in Table I.
Table 111. Reproducibility Tests of Alkaloids Found in Cured Tobacco Sicotine, Yc 1.10 1.10 3.80 3 78 2.53 2.56 2.68 2.63 2.66 2.03 2.94 1.93 3 30 3 26 3.33 3.31 3 03 3 03 2.77 2.74
Nornicotine, V0 0.11 0.09 0.43 0.46 0.25 0.28
0.58 0.59
0.31 0.33 0.79 0.81 0.68 0.67 0.58 0.62 0.81 0.78 0 68 0 66
Total, % 1 21 1.19 4.23 4.24 2.78 2.81 3.26 3.22 2 97 2.96 3.73 3,74 3.98 3.93 3.93 3 93 3.84 3.81 3.4.; 3 40
Standard deviation for nicotine = 0.021. Standard deviation for nornicotine = 0.011.
hbout 1 gram of dry starch is then added and thoroughly mixed with the sample. It is thus dried sufficiently to facilitate transfer to the column. It is poured on the column through a powder funnel and packed in place with a tight-fitting cotton plug, Extraction and Separation of Nicotine and Nornicotine. The sample and column are washed with 200 ml. of hexane under gentle air pressure. The effluent is collected in a 500-ml. Erlenmeyer flask. Sornicotine, which is retained by the column under these conditions, is readily removed by washing the column with 200 ml. of chloroform. The separation and recovery of nicotine and nornicotine added to nonalkaloidal plant material are demonstrated in Table 11. The presence of anabasine and other tobacco alkaloids in either of the extracts may be disregarded for all practical purposes, since they occur in such small amounts. Titration Procedure. The fractions are most conveniently analyzed by titration. Exactly 100 ml. of distilled water is added to the effluent in the collection flask. Two drops of 0.1% bromocresol green indicator and 10 ml. of 0.02 N hydrochloric acid are added. The flask is stoppered, and the mixture is shaken vigorously. The total volume of the water phase will thus be 100 ml. plus the volume of 0.02 N hydrochloric acid added. The phases are allowed t o separate, and a 50-ml. aliquot of the water phase is withdrawn by pipet, and excess hydrochloric acid is titrated with
