by capillary action. At the outset, the level of the fluid should not reach the stick itself. After the front has moved up to 15 to 20 mm., the level is not critical. When the front reaches the top, the stick is removed and the zones are separated by cutting. I n the case of colorless substances, the zones can be detected by the following method. The developed chromatostick, while wet, is painted along its length with S e a t a n (Merck, Darmstadt) emulsion. After 2 minutes, a thin strip of adsorbent can be pulled off with adhesive tape. This strip can be sprayed with a proper chromogenic reagent. The aeight of the mixture to be
separated is dependent on the character, number, and R, values of the components as well as the general purity of the mixture. Forty to 60 mg. of the mixture of three sequirins ( I ) with R, values of 0.68, 0.54, and 0.33 were separated on a single silica gel stick 20 mm. in diameter and 180 mm. in length. Experience has shown that this is the optimum column dimension. Deviations from these proportions led to density gradients and consequently to curved rather than plane zone boundaries. Using multiple holders, batteries of sticks can be developed and larger amounts separated a t the same time. I n the separation of sequirins, the de-
tection of the zones was easy, because slight oxidation resulted in dark, distinct rings, and their positions agreed with the R, values of the spots on a thin layer plate developed under the same conditions. LITERATURE CITED
Balogh, B., Anderson, A . B., Report on Sequirins, 144th Meeting, ACS, LOBAngeles, 1963. (2) Miller, J. M., Kirschner, J. G., ANAL.CHEM.23, 428 (1951). BELABALOGH Forest Products Laboratory University of California Richmond, Calif. (1)
.
Improved Method for the Determination of Benzola]pyrene in Cigarette Smoke Condensate SIR: The appearance of the report to the C . S. Surgeon General entitled “Smoking and Health” (1%) has increased interest in the composition of tobacco smoke. Undoubtedly, many workers will now be studying ways to reduce the benzo [alpyrene content of cigarette smoke. Obviously, such studies will require a reliable analytical method. The low levels of this hydrocarbon in smoke make determination difficult ( I O ) and, in our experience, previously published analytical methods (2, 6 , 7 , I S ) for benzo[a]pyrene are not routinely suitable for several reasons. First, the methods are time-consuming and tedious because of repetitive chromatographic separations and solvent removal steps (4, 7 , I S ) . Second, some of the techniques show poor reproducibility in the pattern of column chromatographic elution (4, 6, 7 ) which is reflected in the ultimate analytical values obtained: in fact, no values are frequently obtained, possibly because of escessive diffusion through the column. The wide range of values reported in the literature (4, 7 , 12, I S ) may be further evidence of poor reproducibility. Third, many of the methods require relatively specialized equipment for spectrophotofluorometric (1, 4 ) or radiochemical determination (6, 7 , I I ) which may be unavailable in many laboratories initiating work in smoke composition. The present method overcomes many of these objection’s and is based on the following steps: single-column chromatographic separation of the nitromethane-soluble neutral substances of smoke; no more than two thin layer chromatographic separations (TLC) of a selected column eluate; and ultraviolet spectral determination of the eluate from the TLC. The column adsorbent is silicic acid which is more highly activated than the partially deactivated
preparations previously used (6, 7 ) . The latter do not give a reproducible elution pattern, thus necessitating extensive screening of all eluted fractions by a tedious sequence of paper or thin layer chromatographic separations followed by spectral examination. When highly activated silicic acid serves as adsorbent, column eluates are essentially colorless and can be monitored directly by ultraviolet spectrophotometric readings at 365 and 384 mp. (5) to detect the presence of benzo [alpyrene, a feature not possible with adsorbents previously described ; in fact, monitoring has not been necessary if smoking conditions and other variables are held rigidly constant. Thin layer chromatographic separation (8) of the chromatographic fraction containing the benzo [alpyrene is more convenient and much faster than the paper chromatographie methods usually employed ( 6 , 7 , I S ) . After one TLC separation, or t n o when further purification is deemed necessary, the characteristic ultraviolet absorbing peaks for benzo[a]pyrene at 365 and 384 mp are definitive, and quantitative measurements may be made (5) easily on eluates from the plates. Although such TLC eluates apparently contain impurities, the latter do not interfere with the analysis; in this respect, the modification is no different from other technique.. . Experiments in which authentic benzo [alpyrene was added to smoke condensate showed an overall recovery of more than SO%, which is comparable if not superior to other published methods. Five replicate analyses of a commercial king-size nonfilter cigarette gave values of 1.5) 1.0, 1.0, 1.0, and 1.2 pg. per 100 cigarettes (uncorrected for losses). Although variable, these values are an improvement over results previously obtained. Analvsis of a sample
requires about 16 hourb compared to 48 hours for other methods ( I O ) involving multiple column and paper chromatographic separations. The method results in values for isolated benzo [ a ] pyrene rather than in absolute value- for benzo [alpyrene in cigarette smoke. Preliminary experiments indicate that the minimum number of cigarertes required to obtain an analytical value by the above method (about 250) may be considerably reduced (to less than 50) by using spectrophotofluorometric instead of ultraviolet spectral determination because ‘of the increased sensitivity. EXPERIMENTAL
Two hundred fifty cigarettes are smoked on an automatic smoking machine. The condensate is collected in traps cooled in dry ice-acetone and the acids and bases are removed from the condensate as previously described ( 3 ) . The residue from the entire neutral fraction is partitioned between cyclohexane (120 ml.) and 4 : 1 niethanolwater (40 ml.), The cyclohexane solution is then extract,ed ( 7 ) with nitromethane (five times, 40 ml. each). The solvent is removed from the nitromethane solution by evaporat,ion in vacuo. The residue is dissolved in t,he minimum amount of hexane and this solut,ion is added to a column (22-mm. 0.d.) containing 50 grams of highly activated Mallinckrodt analytical reagent silicic acid ( I OO-ineshl. (The silicic acid is prepared by activating the methanol-washed adsorbent for 16 hours a t 170” C., slurrying the adwrbent‘ in chloroform, pouring the durry into the column, and washing the column contents with hexane until free of chloroform.) The column ir then eluted with the following aeries of wlvents (200 ml. each): hexane; 99:l hexane-benzene (v. ,‘v.); 9 5 5 hesanebenzene; 90: 10 hex an^-benzenc: and 80 :20 hexane-benzene. Alinmt ali of VOL. 36,
NO, 13, DECEMBER 1964
2499
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 was maintained at 25" 1' C. during 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
