In this case Equation 1 is modified to the following which is valid after the equivalence point: C(V - V,) 2.3RT E = E, - 2.3RT log logy E. (3) Vo F and thus :
+
+v
(Vo
+ v>10-"&
=
10-(Eo+Ej)&
x y x C ( V - V,) (4)
The extrapolation of the straight line described by the experimental points [ V,(V,+ V l O intercepts the abscissa for a value V = V,.
.&]
DISCUSSION Fluoride concentration may be directly determined by adding a sodium fluoride solution and by using Gran's plot to locate the equivalence point. It is assumed that the liquid junction potential and activity coefficient of the fluoride ion do not change appreciably under the conditions of the experiment. If the solution contains only fluoride ions in the microgram per milliliter range, then the small additions of fluoride solution have little effect on these values. In more general cases, however, it is necessary to have the sample and the
standard fluoride solution at the same ionic strength; this goal is reached by making both solutions 1M in any salt such as NaC104 or N a N 0 3 or by using an acetate buffer. The procedure described is more accurate than the direct measurement of the electrode potential and of the other suggested procedures; it is carried out quite rapidly because no slow equilibria are involved when a fluoride solution is added. It can be used to measure fluoride concentration as low as lO-5-lO-6M which corresponds almost to the sensitivity of the fluoride electrode. In this range fluoride cannot be determined by titration with La3+ or Th4+, which are the most accurate and sensitive titration procedures. It is noteworthy to mention that the response of the membrane electrodes is more rapid when going from dilute to concentrated solutions than in the other direction. The discussions of this paper have been concerned with the fluoride electrode; they may be applied to any selective electrode providing a linear relationship exists between electrode response and logarithm of the concentration. RECEIVED for review October 9, 1968. Accepted December 16, 1968.
AIDS FOR ANALYTICAL CHEMISTS I
I
Qualitative Thin-Layer Chromatography of Some Irritants William D. Ludemann, Martin H. Stutz, and Samuel Sass Chemical Research Laboratory, Edgewood Arsenal, Md. 21010
THISREPORT describes a method developed for the qualitative analysis (and identification) of some irritant agents used at one time or another by military and law enforcement agencies. These agents include a-bromobenzyl cyanide (brombenzylcyanide, CA), o-chlorobenzalmalononitrile (CS), a-chloroacetophenone (CN), diphenylaminechloroarsine (DM), and diphenylcyanoarsine (DC). All are considered effective irritants. As there are no published methods for the identification of these compounds as a group of chemical agents, a need existed for such a method. This is especially true today with the increasing use of chemicals in combating crime, preventing personal attacks, and in controlling civil unrest, where one (or a combination) of these agents might be used for riot control. Also included here are qualitative thin-layer chromatographic procedures for the analysis of individual irritant samples which allow detection of the most common impurities associated with the compounds. EXPERIMENTAL Adsorbents and Equipment. Adsorbents used were silica gel-G (Merck-Brinkmann Instruments, Inc.) and acid alumina (Woelm-Alupharm Chemicals). The binder used with the acid alumina adsorbent was Ultracal30 (a modified calcium sulfate, United States Gypsum Corp.). Desaga thin-layer chromatographic apparatus, including a variable thickness applicator (Brinkmann Instruments, Inc.), was used to apply the adsorbents to standard (50 X 200 mm) glass plates in thicknesses of either 250 or 500 p . The chromatoplates were prepared in the usual manner (after Stahl). Slurry compositions used in the chromatoplate preparation are shown in Table I. Only the neutral silica gel-G plates were activated; these were heated at 105 to 110 "C for 30 minutes
and stored over silica gel (desiccant). All plates were allowed to equilibrate over silica gel overnight before use. Standard Irritants. Irritant samples were obtained from various internal agencies located at Edgewood Arsenal. Physical Properties of Irritants CA, brownish liquid, b.p. decomposes at 242 "C CS, white to cream solid, m.p. 93-95 "C CN, pale yellow crystals, m.p. 54-55 "C DM, yellow to green solid, m.p. 195 "C DC, white to pink solid, m.p. 30 "C Detection Reagents. o-Dianisidine (Eastman Organic Chemicals), Technical grade, 0.1 % solution in ethyl alcohol. 4-(4'-Nitrobenzy1)pyridine (K and K Laboratories), 5 % solution in acetone. Quinone (Eastman Organic Chemicals), White Label, 0.5 % solution in methyl alcohol. Sodium perchlorate, 5 solution in aqueous potassium acid phthalate (92.11 mg/100 ml of water), adjusted to pH 5.0 with 1N sodium hydroxide. Iodine, resublimed. m-Dinitrobenzene (Eastman Organic Chemicals), White Label, 1 % solution in ethyl alcohol. Sodium hydroxide (Baker), c.P.,20% aqueous solution. Cupric acetate (Baker), c.P.,0.1% solution in water containing one drop of acetic acid per 50 ml. PROCEDURES Sample aliquots are applied to the plates by conventional means in amounts of 5 to 50 pg per spot on the 250-11 layers and 50 to 100 pg per spot on the 500-p layers. The chromatoplates are developed in solvent-saturated chambers (lined with filter paper) at a room temperature of 25 f 2 "C. On comVOL. 41, NO. 4, APRIL 1969
679
____
Plate Acid alumina Acid alumina Basic silica gel-G Silica gel-G Silica gel-G
~~
~
Table I. Slurry Compositions Used for Chromatoplate heparation Adsorbent Binder Adsorbent layer thickness, p weight, g weight, g 250 60 6 500 138 13.8 250 30 30 250 60 500
Slurrying solvent Solvent Volume, ml Water 45 Water 92 NaOH, 1 N 60 Water 60 Water 115
Table 11. Composite of CA, CS, and CN. DM and DC Included for Comparison
Compound a-Bromobenzylcyanide (CA) o-chlorobenzalmalononitrile (CS) a-Chloroacetophenone (CN) Diphenylcyanoarsine (DC) Diphenylaminechloroarsine and its oxide (DM)
Rf
(Adsorbent, acid alumina; solvent, 5% chloroform in benzene) 4-(4’-Nitrobenzyl) Quinone NaOH, 5% pyridine
0.65
Yellow
Darker yellow
Red-violet
-
0.55
Blue
Reddish spot, blue ring
Yellow-orange
-
0.50
-
Brownish
Blue
-
0.30
-
-
-
Rose
0.00
-
-
-
Orange
pletion of development (100 mm), the plates are air-dried before further treatment. CA, CS, and CN (dissolved in chloroform or carbon tetrachloride) are separated from one another on an adsorbent layer of acid alumina, using a mixture of 5 % chloroform in benzene. After the developed chromatoplate is air-dried, the spots are first visualized with a spray of methanolic quinone and they are noted and marked (CN gives no color under these conditions). On subsequent spraying with the aqueous sodium hydroxide, all three compounds are visualized. Further confirmation is obtained on the same plate with sprays of 4-(4’-nitrobenzy1)pyridine and buffered sodium perchlorate. CA shows an immediate coloration. The plate is heated at 105 ‘C for 10 min., cooled to room temperature, and sprayed with piperidine. The color and location of the spots are noted and marked as they appear, since the colors fade. Color can be renewed by a second and third spraying with piperidine (but with less sensitivity). For determining CS, CN, and the impurities associated with the parent compounds, an acid alumina chromatoplate is used. The developing solvent is 25 dichloromethane in benzene. Exposure to iodine vapors visualizes all of the components. A subsequent spray with 4-(4’4trobenzyl)pyridine detects all of the components with the exception of o-chlorobenzaldehyde. For qualitatively evaluating a known CA sample, silica gel-G is used as adsorbent with benzene as the developing solvent. The nitrobenzylpyridine reagent, followed by perchlorate, heat, and piperidine spray will detect all of the significant impurities with the exception of benzylcyanide and benzyl dicyanide. A spray of rn-dinitrobenzene followed by a spray of 20% sodium hydroxide visualizes these two compounds (preferably on a separate chromatoplate). For separating and detecting DM and DC, an adsorbent layer of silica gel-G with a developing solvent of 20 % acetone in chloroform is employed. Visualization of components is achieved by spraying first with a-dianisidine, then with cupric acetate, and finally with 50 sulfuric acid. DM is separated from its impurities on a basic silica gel-G adsorbent layer using 10% ethyl acetate in methanol as a developing solvent. Visualization is as previously described. I n case a sample is completely unknown (except for the fact that it is an irritant), a group of spot tests are performed prior
z
z
680
o-Dianisidine, sulfuric acid
ANALYTICAL CHEMISTRY
to chromatography. The sample is spotted, in triplicate, on a silica gel-G plate. Each individual spot is then treated with one of the reagent systems above-that is, with quinone, with the 4-(4’-nitrobenzyl)pyridine reagents, and with the o-dianisidine reagents. From the results obtained in these spot tests, the chromatographic system (or systems) best suited for the positive identification of the unknown is chosen.
RESULTS AND DISCUSSION CA, CN, and CS were readily separable from one another on an acid alumina plate in conjunction with a developing solvent of 5 % chloroform in benzene. When the plate was sprayed with quinone ( I ) , CA produced a yellow spot and CS a blue spot. The authors found that a subsequent spray with 5 % sodium hydroxide intensified the yellow color for CA, converted the CS spot to a red with a blue ring, and visualized C N as a brown spot. On the same plate, the nitrobenzyl pyridine spray showed CA as a red spot and on subsequent spray with buffered perchlorate, heatingat 105 “C,and spraying the cooled plate with piperidine, CA became red-violet; CS, yellow-orange; and CN, blue. A reduction in sensitivity occurs when the nitrobenzyl pyridine and its series of reagents is applied after the aqueous sodium hydroxide spray. These results including R values are summarized in Table 11. Qualitative separation of CS and CN from their degradation products and impurities was accomplished on acid alumina using 25 % dichloromethane in benzene solution as the developing solvent. Visualization was accomplished by means of the 4-(4’4trobenzyl)pyridine reagent system. Rf values and color of spots are shown in Table 111. CA was separated from its impurities and degradation products on silica gel-G with benzene as developing solvent. Spots were initially visualized and noted by using the 4-(4‘4trobenzyl) pyridine reagent system. Benzyl cyanide and benzyldicyanide were subsequently detected on the plate by spraying first with (1) A Silvestri, Defense Development and Engineering Laboratories, Edgewood Arsenal, Md., private communication, Nov. 1967.
z
z
1 ethanolic m-dinitrobenzene and then with 20 sodium hydroxide. For detecting 1 pg or less of these two impurity components, the m-dinitrobenzene-sodium hydroxide sprays were best utilized on a separate chromatogram. The composite information for CS, including Rf and detection color, is shown in Table IV. The above system was also useful for preparative purification of CA when a 1-mm thickness adsorbent layer was employed(2). D M and D C in fresh, anhydrous methanol solutions were separable on silica gel-G using 20% acetone in chloroform. Spots were detected at a lower limit of 10 pg using sprays of o-dianisidine and cupric acetate followed by sulfuric acid. DM and D M oxide were separable on basic silica gel-G employing 10% ethyl acetate in methanol as the developing solvent. The same detection reagents were applicable to this system. If the methanol solutions of D M were not chilled or the solutions were older than 10-15 min. only the DM oxide was found. These results are summarized in Table V. DM and D C are also separated from CA, CS, and C N in the acid alumina-5 chloroform in benzene system. These results are included in Table 11. For the identification of irritant, it was preferable to collect vapor or aerosol samples, and subsequently dissolve or extract with ethanol. Extraction was both from aerosol filters or contaminated cloth. CA reacted with dry alcohol (10 to 20% per day under ambient conditions); therefore solutions should be kept reasonably fresh. Numerous thin-layer chromatographic systems were investigated before the methods described above were selected. In addition to the adsorbents used in the selected methods, the following were also investigated for possible use as chromatographic media to separate the irritants: neutral cellulose, basic cellulose, silver nitrate-impregnated silica gel-G, cellulose : silica gel-G mixtures, porous glass, and basic alumina. A large number of individual solvents and solvent mixtures were tested as developing solvents with the above adsorbents. This extensive test program was necessitated by the large difference in the polarities of the two groups of compounds to be separated. Although emphasis has been placed on standard thin layers (250 p ) in this report, the same systems were successfully scaled up to thicker layers (500 p and greater) for preparative chromatography. In these instances, preparative samples were obtained for use as colorimetric and infrared standards (except diphenylaminechloroarsine). It was also possible to scale down the systems for micro-detection (micro-slides) since all the detection reagents described were sensitive to 1 pg or less of agent. The TLC separations of a-bromobenzyl cyanide, a-chlorobenzalmalononitrile, a-chloroacetophenone, and diphenyl(2) M. H. Stutz, W. D. Ludemann, and
258 (1968).
’
s. Sass, ANAL.CHEM.,40,
~ _ _ _ _ _ _ _ _
Table 111. Composite of CAYCS, CN and Some Impurities (Adsorbent, acid alumina; solvent, 25% dichloromethane in benzene) Compound
Rf
0.71 0.58 0.53 0.45 0.29
o-Chlorobenzaldehyde a-Bromobenzylcyanide o-Chlorobenzalmalononitrile
a-Chloroacetophenone Malononitrile Acetophenone a Detected with iodine vapors.
0.00
4-(4’-Nitrobenzyl) pyridine a
Red-violet Yellow-orange Blue Yellow Yellow
Table IV. Composite of CA and Its Impurities (Adsorbent, silica gel-G; solvent, benzene) Compound
Rf
Benzylbromide 0.73 Dibrombenzylcyanide 0.67 E-Bromobenzylcyanide 0.52 Unknown 0.43 0.40 Benzyldicyanide Benzylcyanide 0.29 Unknown 0.11 Unknown 0.04 Phenylbromo0.00 acetamide
4-(4’-Nitrobenzyl) m-Dinitrobenzene pyridine and NaOH Blue Tan Red-violet Violet
-
-
Violet Violet
Violet Blue Blue
-
Table V. Composite of DC, DM, Diphenylaminechloroarsine Oxide, and Diphenylamine o-Dianisidine, Compound RfIa Rmb sulfuric acid Diphenylcyanoarsine 0.65 Rose DiphenylamineOrange chloroarsine 0.35 0.23 Diphenylamine0.08 0.15 Green Diphenylaminechloroarsine oxide 0.38 Orange a System I. Adsorbent, silica gel-G; solvent, 20% acetone in chloroform. b System 11. Adsorbent, basic silica gel-G; solvent, 10% ethyl acetate in methanol.
cyanoarsine were sufficiently clean to allow probable quantitation by spectrophotometric or densitometric procedures.
RECEIVED for review September 16, 1968. Accepted November 27, 1968.
VOL. 41, NO. 4, APRIL 1969
681
