liberating about ‘/3 mole of iodine in a n hour. Styrene oxide apparently shows rapid liberation of a mole of iodine from the epoxide group, and then reduction of the resulting styrene a t its usual moderate rate. Further illustration of the effect of alkyl substitution on the reduction of exocyclic functionality is shown in Figure 6, by a series of benzyl alcohols. In a turnabout of most alcohol reaction rate series, the primary alcohol is the least and the tertiary the most reactive -reflecting the relative ease of reduction of the corresponding alkyl iodides. Referring again to Table XII, a comparison of results for styrene and the almost inert trans-stilbene shows the stabilizing effect of the extra aromatic substitution against HI reduction of the exocyclic double bond. The destabilizing effect of methoxyl substitution can then be seen with dimethoxystilbene, which had moderate reactivity. Anethole also is moderately reactive. AnisoIe. however, is inert, which indicates that iodine liberation by the niethoxy compounds is not due to HI cleavage of the ether groups. LITERATURE CITED
(1) Barnard, D., Hargrave, K. R., Anal.
Chzm. Acta 5,476 (1951). (2) Bruschweiler, H., Minckoff, G. J., Zbid., 12, 186 (1953). (3) Bukata, S. W.,Zabrocki, L. L., McLaughlin, AI. F., ANAL.C m v . 35, 885 (1963).
(4) Dastur, N. N., Lea, C. H., Analyst 66, 90 (1941). ( 5 j -Dav&,’- A. G., “Organic Peroxides,” Chap. 14, Butterworths, London, 1961. (6) Dickey, F. H., Raley, J. H., Rust F. F., Treseder, R. S., Vaughan, W. E., Znd. Eng. Chem. 41, 1673 (1949). (7) Dubouloz, P., Fondarai, J., Laurent, J., Marville, R.,Anal. Chim. Acta 15, 84 (1956). (8) Franzke, C., 2. Lebensm. Untersuch. Forsch. 103, 108 (1956). (9) Goddu, R. F., Advan. Anal. Chem. and Instr. 406-9 (1960). (lO).Goddu, R. F., unpublished work, this laborator (11) Hawkins, G. E., “Organic Peroxides,” Chap. 11, Van Nostrand, Princeton, N. J., 1961. (12) Heaton, F. W., Uri, N. J.,Sci. Food Agr. 9, 781 (1958). (13) Hock, H., Kropf, H., Chem. Ber. 92, 1115 (1959). (14) Hock, H., Schrader, O., BrennsbffChem. 18, 6 (1937). (15) Kokatnur, V. R., Jelling, M., J . Am. Chem. SOC.63, 1432 (1941). (16) Kolthoff, I. M., Medalia, A. I., ANAL. CHEM. 23, 595 (1951). (17) Kuta, E. J., Quackenbush, F. W., Ibid., 32, 1069 (1960). (18) Mair, R. D., Bobst, J. K., unpublished work, this laboratory. (19) Martin, A. J., “Organic Analysis," Vol. 4, pp. 1-64 John Mitchel, Jr., ed., Interscience, New York, 1960. (20) Mayo, F. R.,Walling, C., Chem. Revs. 27, 351 (1940). (21) Moss, M. L., unpublished work, this laboratory. (22) Nozaki, K., IND. ENG.CHEM.,ANAL. ED. 18, 583 (1946). (23) Pobiner, H., ANAL. CHEW 33, 1423 (1961). (24).Quackenbush, F. W., Kyta, E. J., Division of Analytical Chemistry, paper
&.
Adaptation of a Galvanic of Oxygen HARRY LIPNER,
L. R. WITHERSPOON, and V. C.
Cell
35, 142nd Meeting, Am. Chern. Soc., Atlantic City, N. J., September 1962. (25) Roth, H., Schuster, P., Mz’krochim. Acta 6,840 (1957). (26) Royals, ,?, E., “Advanced Or anic Chemistry, p. 369, Prentice-hall, Englewood Cliffs, S. J., J954. (27) Ryland, Ada L., Division of Analytical Chemistry, paper 33, 142nd Meeting, Am. Chem. SOC., Atlantic City, N. J., September 1962. (28) Siggia, S., ANAL. CHEM. 19, 872 (1947). (29) Silbert, L. S., J . Am. Oil Chemists’ SOC.39,480 (1962). (30)Silbert, L. S., Swern, D., AXAL. CHEM.30, 385, (1958). (31) Silbert, L. S., Ritnaur, L. P., Swern, D., Ricciuti, C., J . Am. Chem. SOC.81, 3244 (1959). (32) Slover, H. T., Dugan, L. R., J. Am. Oil Chemists’ SOC.35,350 (1958). ( 3 3 ) Sorge, G., Ueberreiter, K., Angew. Chem. 68, 352, 486 (1956). (34) Sully, B. D., Analyst 79, 86 (1954) (35) Swern, D., “Primary. Products of Olefinic Autoxidations” in “Autoxidation and Antioxidants,” Vol. I. W. 0 Lundberg, ed., Interscience, New York, 1961. (36) Vaughan, W. E., Rust, F. F., U. S. Patent 2,403,771 (July 9, 1946). (37) Wagner, C. D., Smith, R. H., Peters, E. D., IND.EKG. CHEM., ANAL.ED. 19, 976 (1947). (38) A’ibaut, J. P., VanLeeuwen, H. B., van der Wal, B., Rec. Trav. Chim. P U ~ S - B 73, U S 1033 (1954). (39) Wolfe, W. C., ANAL.CHEM.34, 1328 (1962). RECEIVEDfor review June 6, 1963. Accepted October 10, 1963. Division of Analytical Chemistry, 142nd Meeting, ACS, Atlantic City, N. J., September 1962.
for Microanalysis
CHAMPEAUX
Department of Biological Sciences and Department o f Chemistry, The Florida State University, Tallahassee, flu.
b A galvanic cell is described which may b e used to measure oxygen content or rate of change in oxygen content on the order of 7 X 10“ pl. oxygen/ml. sample/minute. Evidence is presented for the reliability and accuracy of this cell and advantages over previous methods are discussed.
M
m T H o D s have been employed for the measurement of oxygen change in biological systems. The Winkler titration method ( I ) , manometric methods (13), and variations of the Davies and Brink electrode method (4) have been used to determine oxygen change. The application of galvanic cells to the measurement of oxygen change in biological systems is not novel (3, 6, 9-12), Galvanic cell methods based on the ilg/KOH/Pb
204
ANY
ANALYTICAL CHEMISTRY
chemistry have also been employed for the measurement of oxygen content (d, 5, 7 , 8). We have introduced a bucking potential circuit in series with the galvanic cell designed by Mancy, Okun, and Reilley (8) and can measure oxygen changes on the order of 7 X 10-5 pl. O2 per milliliter of solution per minute. Our galvanic cell was calibrated to report information in terms of microliters of oxygen rather than voltage produced. EXPERIMENTAL
Apparatus. The galvanic cell (Figure 1) consists of a silver cathode set into plastic in the center ‘of the cell and surrounded by a lead ring anode. T h e electrodes are separated by a distance of 0.2 mm. The tip of the cell is covered with a 1N KOH saturated disk of lens paper which is
then covered with a polyethylene film (W. P. Ballard & Co., Birmingham, Ala.) 0.5 mil (12.7 microns) thick secured by a n 0 ring. The galvanic cell was constructed as described by Mancy, Okun, and Reilley (8). Oxygen present in solution diffuses through the polyethylene membrane and is reduced at the silver cathode. The lead electrode reaction is the oxidation of lead, forming lead oxide complex. The resultant flow of electrons is from lead t o silver. The supporting electrolyte used is 1N KOH, because it is highly conductive, but residual current is small in the absence of oxygen. The diffusion current generated is determined by the rate of oxygen diffusion through the polyethylene membrane which depends on the concentration of dissolved oxygen in the reaction mixture. The system is highly temperature dependent and efficient stirring of the solution is necessary for precise determination of contained oxygen.
RECORDER
CELL
I WPRD'S 131 OP L A STlC IO K /
i
CEL.
II
G L A S S ENCLOSED STEEL BAR STIRRER
II
Figure 2. Circuit diagram showing the introduction of a bucking potential opposed to the potential drop across the current measuring resistor
EVALUATION OF CELLPERFORMANCE. To confirm that the amount of oxygen present in a sample is directly pro-
FILM
portional to the diffusion current generated by the galvanic cell, a series of samples was run in which the rate of LEHS PAPER DOPA oxidation of tyrosinase was SATURATED IN cm I N. KOH measured by the cell. *A plot of initial rate of oxidation us. enzyme concenA 8 tration is a straight line since substrates Figure 1A. Galvanic cell, with slight modification from that described b y are not limiting. Mancy, Okun, and Reilley (8) The millivolt change is directly re6. Cell housing. Sanples are introduced into the jacketed tube in which is shown lated to the oxygen consumed by the the galvanic cell system which is in turn directly dependent on the enzyme concentration. Figure 3 is a plot of initial rate of oxidaThe putential de\.eloped across the for ouygen using the Kinkler method tion us. milliliters of enzyme in a fixed current measuririg resistor is opposed volume and because this plot has a and the equivalence of oxygen conwith a \oltage divider across 1.3-volt constant slope, it demonstrates the centration to voltage generated was mercury cell (Figure 2 ) . (This allows reliability of the galvanic cell over a determined. small changes in the generated current to DOPA\ oXID.4TIOK O F TYROSISASE. range of oxygen concentrations. Even be detected.) X 10-mv. full scale dethough freshly prepared galvanic cells A crude mushroom tyrosinase solution, flection recorder is used with this were used for each run, similar values 3 X DOPA, and 2.2 X 10-2.1~ system and a 0.l-niv. change in the IR were obtained for rate dependence on NaH,P04 adjusted to pH 7.0 were drop is readily detected. The cell is enzyme concentration. This demonprepared. Mixtures of these three housed in a jacketed 1 ube through which solutions were prepared, varying the it-ate1 a t 30" C. is rapidly circulated. amount of enzyme but always adding -1magnetic stirrer is provided. f 0.2 ml. of DOPA and adjusting to a Procedure. CALLBRATION OF THE final volume of 2.0 ml. with NaH2P04. C E L L . An oxygen calibration curve A fresh cell n-as prepared each day to was prepared by using samples of 35 Slope. 100 avoid complications due to adsorption water containing vrtried oxygen con40 of materials on the membrane. centrationq. An aliquot was analyzed
1
260
30t 1
T
,
240
220
180
0.066ulO,/cc./mv
0
4
120
Figure 3. Determination of equivalence of measured potential to oxygen present
::f
Equivalence for our cell was 1 mv./0.066 pl. oxygen/ml. sample ml.Enzyme (Tyrosinasel
20
Figure 4.
uI O,/ml.HIO
Reliability of galvanic cell
Comparison of slopes obtained a t different times using different ranges of enzyme concenfration demonstrates reproducibility obtainable
VOL. 36, NO. 1, JANUARY 1964
205
strates that each fresh galvanic cell is equivalent and that runs made with the same cell a t different times are comparable. DISCUSSION
A modification of the galvanic cell of Mancy, Okun, and Reilley has been described which may be successfully employed in the measurement of oxygen consumption a t the micro level. This cell has been calibrated so that the exact oxygen content corresponding t o the current generated by the cell is known. Figure 4 illustrates the calibration for our cell. A 1-mv. change in loaded cell voltage is equivalent to a change of 0.066 111. of oxygen per milliliter of solution per minute. The reproducibility of data has been confirmed over a period of time and the precision of the instrument is well within experimental error. The advantages of this galvanic cell are many and varied, The cell provides almost immediate evaluation of oxygen content, allowing many samples t o be run in a
short time. Continuous monitoring of a reaction is possible. The addition of reactants to the reaction chamber may be done a t any time during a run. In contrast with the oxygen electrode, no external source of potential is needed and no amplification is needed, rendering expensive or complex electronic equipment unnecessary. The method described is easily as sensitive as any used in the quantative evaluation of oxygen or oxygen consumption. ACKNOWLEDGMENT
We express appreciation to Earl Frieden, Department of Chemistry, Florida State University, for his kind donation of crude tyrosinase. LITERATURE CITED
(1) American Public Health Association, “Standard Methods for the Examination of Water and Wastewater,” 11th
Ed., p. 309, American Public Health Association, New York, 1960. (2) Bates, D. V., Harkness, E. V., Can. J . Biochem. Physiol. 39, 991-9 (1961).
(3) Damaschke, K., Rothvuhr, L., Toat, F., Biochem. 2. 326, 424-32 (1955). (4) Davies, P. W., Brink, F., Jr., Rev. Sci. Instr. 13, 524-33 (1942). (5) Dewey, D. L., Cray, L. H., J . Polarog. SOC.7, NO. 1, 15-25 (1961). (6) Ewald, G., Bruchmann, E. E., Biochem. 2. 324, 156-9 (1953). (7) Hersch, P., Chim. Anal. (Paris) 41, 189-97 (1959). ( 8 ) Mancy, K. H., Okun, D. A., Reilley, C. N., J . Electroanal. Chem. 4, 65-92 (1962). (9) Tpdt, F., Angew. Chem. 67, 266-70 (1935). (10) Todt, F., Cherniker-Ztg. 83, 483-90 (1959). (11) Todt, F., et al., Z. Naturforsch 9b, 607-1 1 (1954). (12) Todt, F.,Todt, €1. G., Vom Wasser 20, 72-126 (1953). (13) Umbreit, W. W., Burris, R. H., Stauffer, A. F., “Manometric Techniques,” 3rd ed., Burgess Publishing Company, Minneapolis, 1957. RECEIVEDfor review May 13, 1963. Accepted September 30, 1963. This investigation was supported by PHS research grant 1904 from the XAMBD, Public Health Service, and in part by a contract between the Division of Biology and Medicine, U. S. Atomic Energy Commission, and the Florida State University.
Spectrophotometric Determination of Iron and Aluminum in Silicone Polymers SHIZUO FUJIWARA and HISATAKE NARASAKI Department o f Chemistry, Faculty o f Science, The University o f Tokyo, Bunkyo-ku, Tokyo, Japan
b Small amounts of metals in silicone polymers were determined by a wetashing procedure. The polymers were decomposed by heating with concentrated sulfuric acid. The organic residue was then oxidized b y 1 drop of nitric acid. The insoluble residue, being mostly composed of silica, was separated from solution b y filtration. Iron and aluminum in both the residue and the filtrate were determined spectrophotometrically. Iron was determined using 2,2’-bipyridyl and aluminum was determined b y the 8quinolinol-benzene extraction method.
T
are essential for the control of both production processes and properties of polymers. We have previously investigated the analysis of trace impurities in certain polymers (8, 4, 18). The presence of trace amounts of metals bears a n important relationship to the electrical and chemical properties of silicone polymers. Although the determination of small amounts of metals in silicone polymers is not contained in the literature, processes RACE ANALYSES
206
0
ANALYTICAL CHEMISTRY
for the analysis of silica in silicone polymers have been published (9, 16). The decomposition of silicone polymers may be accomplished by acid digestion using either a mixture of sulfuric and nitric acids (6), sulfuric and perchloric acids (la), perchloric and nitric acids (6), or other acid mixtures; or by fusion with sodium peroxide in a Parr peroxide bomb (IO). Sodium peroxide often contains a considerable quantity of metallic impurities and is, therefore, not a dependable reagent to use for the determination of traces of metals usually found in these polymers. I n this laboratory, sulfuric acid and 1 drop of nitric acid were used for the decomposition of silicone polymers. [In a sulfuric acid medium the polymers may be broken up into fragments such as silyl sulfate and dissolved (I&.] Then the solution was diluted with water and filtered. The separated residue was ignited and weighed, and subsequently treated by the method of rapid analysis of silicate rocks and minerals (16). The residue part (solution A) and the sulfuric acid soluble part (solution B) were analyzed separately. A procedure
utilizing 2,2’-bipyridyl was used for the determination of iron ( I I ) , and an 8-quinolinol-benzene extraction procedure was used for aluminum (7,8). An attempt was made to ignite the polymer in a Parr oxygen bomb ( I ) , but complete ignition could not be attained. EXPERIMENTAL
Apparatus and Reagents. Hirams, Model I1 B photoelectric spectrophotometer with 1-cm. and 2-cm. cells. T6a Denpa KGgy6, Model HM-5A p H meter. 8-Quinolinol, 1%. Dissolve 1 gram of 8-quinolinol in 3 ml. of glacial acetic acid with warming and dilute to 100 ml. with water. Prepare freshly as required. Calibrations. IROX.By means of a pipet introduce exactly 0, 1, 2, 3, 4, and 5 ml. of the standard iron solution (10 pg. of iron per ml.) into separate 50-ml. volumetric flasks. Add 10 ml. of 0.5N hydrochloric acid and follow the procedure given for iron beginning with the addition of hydroxylamine hydrochloride. ALUMINUM.By means of a micropipet introduce exactly 0, 0.3, 0.6, 0.9, 1.2, and 1.5 ml. of the standard alu-
