the benzo[a]pyrene is found in the 90: 10 hexane-benzene eluate (in fractions 14 to 16 when 50 ml. fractions are collected) and is easily detectable by the characteristic absorption maxima at 365 and 384 mp. Occasionally trace amounts are detectable in the first 50 ml. of the 80 :20 hexane-benzene eluate. By collecting 50-ml. fractions, other polynuclear hydrocarbons,-e,g. benz[alanthracene-can probably be isolated and determined, although such determinations have not been extensively studied at present. The benzo [alpyrene fractions are pooled, concentrated, and applied as a band on an 8 x 8-inch glass plate coated with silica gel G. The plate is developed in a solvent system of 19:l n-pentane-ther (9). After development, the band corresponding to benzo [alpyrene is located by examining the plate under ultraviolet light. The band is scraped from the plate, and washed three times (4 mi. each) with methanol by slurrying and decantation. (Occasionally it may
be necessary to repeat the thin layer chromatography with the residue from the pooled methanol washes to improve the spectrum.) The final pooled methanol solution is evaporated to dryness, the residue is dissolved in cyclohexane, and the solution is read a t 384 mp (5). More than two TLC on the same or different substrates (8, 9) do little to improve the spectrum. LITERATURE CITED
(1) Barkemeyer, H., Beitr. Tabak-Forsch. 1, KO.9, 325 (1962). (2) Bentley, H. R., Burgan, J. G., Analyst 83, 442 (1958). (3) Burdick, D., Stedman, R. L., Tobacco Sci. 7, 113 (1963). 14) Cardon. S . Z.. Alvord. E. T.. Rand. H. J., Hitchcock, R., brit. J.' Cance; 10, 485 (1956). (5) Cooper, R. L., Analyst 79, 573 (1354). (6) Hoffmann, D., Wynder, E. L., ANAL. CHEM.32, 295 (1960). 17) . , Hoffmann. D.. Wvnder. E. L.. Cancer 13, io62 (ibsoj. " (8) Matsushita, H., Suzuki, Y., Sakabe, \
,
~
H., Bull. Chem. SOC.Japan 36, 1371 (1963). \_.__
(9) Sawicki, E., Stanley, T. W., Elbert,
W. C., Pfaff, J. D., AIVAL.CHEM.36,
497 (1964). (10) Sawicki. E., Chemist-Analust 53. 24 (1964). (11) Scherbak, M., Rice, R. L., de Souza,
J. E., Abstracts, 17th Tobacco Chemists' Research Conference, Montreal, Cannrln ---1
IQFI.?
(12) "Smoking
and Health," Public Health Service Publ. S o . 1103, U. S. Deat. of Health. Education and Welfare. Washington. D. C.. 1964. (13) van DuuGn, B. L., 2. Natl. Cancer Inst. 21, l(1958).
IRWIN SCHMELTZ R. L. STEDMAN W. J. CHAMBERLAIN Eastern Utilization Research and Development Division Agricultural Research Service U. S. Department of Agriculture Philadelphia, Pa. 19118 USE of a commercial product does not constitute endorsement by the U. S. Department of Agriculture over other products of a similar nature.
Direct Potentiometric Determination of Urease Activity SIR: Current methods for determining urease activity involve the determination of the amount of ammonia formed within a given period of time by t h e action of the enzyme on urea. The ammonia is determined by titration (6, 12) or nesslerization (8, 2 1 ) after quenching the reaction. A direct potentiometric method utilizing a cationic sensitive glass electrode to determine the ammonia liberated in this reaction has been developed. This method is more rapid than titration or nesslerization, and no isolation of ammonia is involved. EXPERIMENTAL
All potentiometric measurements were made with a Corning model 12 p H meter on the expanded scale. The Beckman 39137 cationic sensitive glass electrode and the Corning fiber junction calomel electrode were employed. The meter output was recorded on a Heath EUW 20-A recorder. The temperature 1' C. during was maintained at 25" the course of these experiments, and the system under investigation was
*
stirred by means of a rotating magnetic stirrer. All solutions were prepared from reagent grade chemicals. Commercial urease powder was obtained from local laboratory supply houses. Preparation of Calibration Curve. A series of solutions from 5 X lop2 to 5 x 10-5M in ammonium sulfate maintained a t pH 7.0 by a 1 X 10-'M trishydroxymethyl amino methane (THAM) buffer was prepared. Potentiometric measurements were made on these solutions. A plot of log [NH4+].against the observed potential in millivolts gave a straight line of the form:
+
E = 50.1 log ["(+I 288.3 (1) where E is the potential in millivolts. Recovery of Ammonia. To evalua t e the accuracy with which ammonia could be determined in the presence of urease, a n d to determine the speed with which the electrode responded t o changes in the ammonium ion concentration, 0.5-ml. increments of 5 X 10-1M ammonium sulfate were injected into exactly 60 ml. of the THAM buffer containing a weighed amount of urease powder. The change in poten-
Table 1. Recovery of Ammonia in Presence of Urease Urease, 0.0105 gram Urease, 0.0337 gram I+&"[ added [NHa+]found [NHl+] added ["I+] found 8 . 7 6 x 10-3 8.85 x 10-3 8.92 x 10-3 8.76 X 1.74 2.59 3.42 6.63 9.64 1.25
2500
X X X X X lov2 X 10-1
1.80 2.65 3.50 6.66 9.70 1.31
X X X X X X 10-1
ANALYTICAL CHEMISTRY
1.74 X 2.59 X 3.42 x 4.24 X 5.05 X 5.84 X 6.62 X
1.74 2.62 3.58 4.33 5.21 6.02 6.58
x x x 10-2 X X X X
tial of the indicator electrode was immediate and constant. The ammonium ion concentration was then calculated from these potentiometric measurements with the above equation. These values are compared to the theoretical values in Table I. Determination of Urease Activity. A weighed amount of urease powder was dissolved in exactly 60 ml. of T H A M buffer. T h e electrodes were immersed in the solution and the potential was recorded. Exactly 10 ml. of 0.5M urea solution was added. The ammonia liberated by the action of the enzyme was determined from the potential value observed 5 minutes after the addition of the urea. The results of several assays are listed in Table 11. ~~
Table II. Results of Potentiometric Urease Activity Determinations Activity, mmoles Ammonia NH, Urease formed formed/ Samtaken, in 5 min., urease, Ple 1 1 1
gram
mmole
gram
0.0338 0.0334 0.0078
1.79 1.77 0.421
53.0 53.0 54.0
2 2
0.0084 0.0115
0.192 0,268
22.8 23.2
3 3 3 3 3 3
0.0182 0.0107 0.0188 0.0144 0.0224 0.0118
0.571 0.388 0.629 0.536 0.777 0.393
31.4 37.1 33.5 36.9 34.7 33.3
DISCUSSION
Many investigators (1-4, 6, 7‘) have reported on the linearity of the responbe of the cationic sensitive glass electrode to the logarithm of the concentrations of monovalent cations of Li, Iia, K, NHs, Rb, Cs, and Ag. Accuracy, as evaluated in the ammonia recovery study, is within 5% of the theoretical amount of ammonia present. The determination of urease activity is basically the same as existing methods; but the determination of the ammonia liberated is by potentiometric measurement. This leads to a more rapid method, and there is no interference from the enzyme, substrate, or buffer. I n addition, there is no need
to quench the reaction in order to make the potentiometric measurement. Hence, a continuous measure of the ammonia liberation is available. This procedure may be of value in re-evaluating previous work (9, 10) on the rate of the urease catalyzed hydrolysis of urea. LITERATURE CITED
(1) Beckman Instruments Inc., Fullerton, Calif., Bull. 7017 (1961).
(2) Beckman Instruments Inc., Fullerton, Calif., Bull. 741-B (1961). (3) Eisenman, G., Biophys. J., 2, part 2 (supplement), 259 (1957). (4)Eisenman, G., Rudin, D. O., Casdy, J. U., Science 126, 831 (1957). (5) Gorin, G., Fuchs, E., Butler, L., Chopra, S., Hersh, R., Biochem. 1, 911 (1962).
(6) Isard, J., Nature 184, 1616 (1959). (7) Katz, S.,Rechnitz, G., Ann. Meeting of the N. J. Acad. Sci., Glassboro. N. J., 1963. (8) Kistiakowsky, G., Rosenberg, A,, Lumry, R., Thompson, W. E., J . Am. Chem. SOC.74, 5015 (1952). (9) Kistiakowsky, G., Rosenberg, A,, Ibid- . > rn - 5020
Laidler, K., Hoare, J., Ibid., 71, (1?2!399 (1949). (11) Sumner, J., Hand, D., J . Biol. Chem. 76, 149 (1928). (12) Worthington Biochemical Coro..
. Freehold,
s. J., 3.5.1.5 (1963).
I
>
SIDNEY A. KATZ Department of Chemistry Rutgers University Camden, N. J. 08102 FINAXCIAL support from the Rutgers University Research Council is gratefully acknowledged.
Limiting Case for the Difference to Sum Ratio Method in Photoelectric Polarimetry SIR: In a recent paper (S), Kahn, Calhoun, and Ritnauer have shown how the Brice light scattering photometer may be modified for use as a photoelectric polarimeter. They used the difference to the sum ratio method and were able to obtain a standard deviation of ~ t 0 . 0 1or~ better. They also indicated a number of sources of error in this method and correctly called attention to an apparent inconsistency in our treatment of the sensitivity of the method ( 2 , 4). It is the purpose of this paper to analyze the sensitivity in the limiting case as experimentally investigated by Kahn and coworkers, this being the case where the sum of the fixed angle between the optic axes of the polarizer and analyzer, 6, and the optical activity of the sample, a , approaches the value of 90’. Some practical suggestions for photoelectric polarimetry become evident as a result of this analysis. Sensitivity for a = ( r / 2 - 6). In t h e difference to sum ratio method we have
and
R =
2tanBtana
1
+ tanz8tanza
T o obtain the sensitivity, (aR/aa), one may expand Equation 4 as
R
=
2tan6 X
2(1
CY
-
- 1 / 3 t a n 2 0 ) ~ ~ Xn ’a~‘
cosZ(6 R= cos2(6
- a ) -_cos2(6 + _ a) - CY) + cos2(6 + a ) (2)
This equation may be shown to be equivalent to
+ ...
(5)
Clearly the approximation we have previously used ( 4 ) as the sensitivity around the origin-Le., for small values of a
aR/aa
‘v
(6)
2tan6
is valid provided that R may be represented by the linear term in Equation 5 . Equation 6 covers a useful range of 6 values. When the angle, CY, becomes large, particularly when a tends to equal the complement of the angle 6 , then cos(6 - a) -+ sin26 and cos(6
where El and Ez are the energies intercepted by the phototransducer when the fixed angles are plus and minus 6, respectively. For nonscattering and nonabsorbing solutions (1, 6), the conventional form of Malus’ equation may be used so that Equation 1 becomes
(4)
+ a)
+
0
-
so that Equation 2 yields R 1. T o evaluate the sensitivity under these conditions one should consider Equation 3. This yields
aR/aa
=
2sin28 X [cos26 [i
+
+ C O S ~ C U(7) ]
C O S ~ ~ C 1O2 S ~ ~
~It will be seen from Equation 7 that as Q -+
~ / 2 6, then aR/aa
-+
0
i.e., the sensitivity goes to zero. This is in accord with the experimental findings of Kahn, Calhoun, and Witnauer [(S), Figure 51.
Effect of Noise in t h e Region a = n/2 - 6. T h e presence of noise components in this region should have a large effect upon the precision. T h e fact that t h e latter investigators achieved a maximum value of R = 0.870 instead of the theoretical value, R = 1, may be taken as a measure of the noise component when CY = ~ / 2 6. It is questionable whether complete reliance upon calibrations with known substances can be satisfactory in the face of such a sizable noise component and poor sensitivity. For measurements of greatest precision, one should try to keep in that range of 6 and a where only the linear term in Equation 5 is a good approximation. As the latter investigators were careful to indicate, deviation of the observed R from its theoretical value as calculated from Equation 2 may be ascribed in part to such factors as: imperfections in the polarizer and analyzer permitting unpolarized and circular polarized radiation to be intercepted by the transducer and depolarization of the incident polarized radiation caused by scattering and appreciable dark current. All these sources of unattenuated radiation will tend to lower the ratio, R, because it has been shown ( I , 5, 6) that under these conditions one may write
R= Cd(8
-
-
a)
COS'(^
a)
- cos2(6
+ a)
+ COS^(^ + + E , CY)
(8)
Here E , is a function representing the unattenuated energy intercepted by the phototransducer and may be considered a noise component. Examination of Equation 8 shows t,hat neglect of the noise component E , introduces VOL. 36, NO, 13, DECEMBER 1 9 6 4
2501
