Table 11.
Dependence of AF/At upon ADH Concentration [EtOH] = 0.066M, diaphorase = 0.08 unit/ml., resazunn = 6 X 10-M AF/Al, [ADHI, umts/ml. F units/min. 0,000303 0.000606 0.00151 0 . 00606 0,0:303 0.0fioti
0.om 0.040 0.100 0.410 2.00 4.01
acid dehydrogenase, LAD, in concentrations as low as 0.000470 unit per ml. (Table I). This reaction was also first order with respect to diaphorase in concentrations of 0.0004 to 0.080 unit per ml. of solution. and to resazurin (10- to
lO'M). Hence the concentrations of these materials may be easily determined by this technique. Also, the rate of formation of r e s o r u h was proportional t o the NAD+ concentrations in the region 10-'0 to lO'M, thus allowing an easy, direct determination of the quantity of this eoeneyme present. By the use of ethyl alcohol as substrate, the quantity of alcohol dehydrogenase as low as 0.000303 unit per ml. may be determined. Full details on the systems described herein, as well as applicittion to other systems, will be given in an article to follow. LITERATURE CITED
(1) Cshn, R. D., Kaplan, N. O., Levine,
L., Zwilling, E., Sctenee 136,962 (196'2). (2) Guilbnult, G. G., Kramer, D. N., ANAL.CHEM.36, 409 (1964).
(3) Kagi, J., Vallee, B. L., J. Bid. Chhem. 235, :X88 (1960). (4) Kramer, n. N., Guilbnult, G. G., ANAL.CHEM.35, 588 (1963). (5) Ibid., 36, 1662 (19M). (6) Lowry, 0. H., J . Bid. Chem. 173, 677 (1948). (7) Lowry, 0. H., Roberts, N. R., Chang, M., Ibid., 222, 97 (1956). ( 8 ) Lowry, 0. H., Roberta, N. R., Knpphahn, J . I., Ibid., 224,1047 (1957). (9) hlahler, H. R., Sarkar, N. K., Vernon, L. P., Alherty, R. A,, Ibid., 199, 585 (1952). (IO) Udenfriend, S., "Fluorescence Asmy in Biology and Medicine," p. 312, Academic Preaa, New York (196'2).
GEORGE G. GUILBAULT DAVIDN. KRAMER Defensive Research Division Chemical Resertroh and Development Labs. Edgewood Arsenal, Md. RECEIVEDfor review July 17, 1964. Accepted September 22, 1964.
Preparative Analog to Thin Layer Chromatography SIR: In the course of a study of the isolation and separation of sequirins ( I ) , a new preparative analog of thin layer chromatography (TLC) was developed. Self-supporting chromatographic columns, chromatosticks, were prepared from various T L C adsorbent materials. Chromatohars, chromatographic columns not encumbered by a containing envelope, were first prepared in 1951 by Miller and Kirschner (2) who pointed out many of the merits of baresurfaced columns, such as locating resolved zones and separating them by dissection. They employed special techniques to prepare, support, and develop t.heir chromatohars. The available TLC adsorbents are ideal for the preparation of self-supporting columns which can be processed in a manner much simpler than described by Miller and Kirschner.
Figure 1, Removal of o wet stick from the paper-roll (left). Protective holder made from aluminum wire, with the cotton pillow to transmit the developing fluid (right)
Silica gel sticks were prepared from
EXPERIMENTAI
a slurry of 25 grams of silica gel G (Merck, Darmstadt) and 50 ml. of water in a paper cylinder 20 mm. in diameter and 180 mm. in length.
Preparation of Chromatosticks. I n general, the chromatosticks were prepared by pouring a slurry containing suitable proportions of liquid and adsorbent into a cylinder rolled from filterpaper (Whatman No. l ) , and held together by strips of adhesive tape. The cylinder stands in a wooden holder in which the hottom plate contains it circular and perforated recess lined with filter paper. After a short draining period the moist column can he removed, dried, and activated.
After 15 minutes the wet chromatw stick can be removed and put into a supporting holder (Figure 1). The wire holder shown prevents breakage during handling. After being dried overnight at room temperature, and subsequently for 2 hours at 70" C., the stick was activated at 110' C. for 1 hour. Cellulose sticks were prepared from a mixture of 15 grams of MN-cellulose powder 300 G (Mackerey, Nagel & Co., Dueren) and 4.5 grams of silica gel G suspended in 70 ml. of water.
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ANALYTICAL CHEMISTRY
After 40 minutes, the wet stick can be removed and dried for 3 days at room temperature. Polyamide alone resulted in very fragile chromatosticks; therefore it was used in conjunction with silica gel also. Sixteen grams of Polyamide WOELM (M-\$'oelm-Eschwege, Germany) and 4 grams of silica gel G were mixed and suspended in a mixture of 80 ml. of methanol and 8 ml. of water. After draining for 60 minutes, the stick was removed and dried a t room temperature for three days. Separation Technique. The top of the chromatostick is carefully leveled wit,h a razor blade and the solution of the mixture to he separated is applied as spots in different patterns-e.g., diagonally, vertically, or i n concentric circles with a micropipet: The volume of the solution should be sufficient to cover the top a t least three times to produce a uniform layer. Iletween applications, the layer is dried wit,h a fan. In common TLC, the width of the start line is 2 to 4 mm.-i.e., the diameter of the spots. In the chromatosticks the layer containing the mixture is less than 1 mm. t,hick; ttierefore, a very good start line is provided, leading to a sharper separation. After the mixture is applied, the sticks are carefully reversed in their holders and placed layer-end down on cotton pillows (Figure 1) cut from folded cotton fleece. The chromatost,icks, either singly or in groups, are placed in a previously saturated chromatography jar. The pillow uniformly transmits the developing fluid to the end of the stick
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. In 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 Seatan (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. In 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
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