DISCUSSION
Front face optics has been employed for qualitative fluorescence analysis of bijAogically important molecules (8-18). Despite the existence of commercial instruments, that provide (8) D. 9. Bender, E. Sawicki, and R. M. Wilson, Jr., ANAL.CHEM., 36, 1011 (1964). (9) B. Chance and H. Caltscheffsky,J . Biol. Chem., 233,736 (1958). (10) B. Chance, P. Cohen, F. Jobsis, and B. Schoener, Sciertce, 136, 325 (1962). (11) B. Chance, J. R. Williamson, P. Jamieson, and B. Schoener, Biochem. Zeitsch., 341, 357 (1965). (12) L. N. M. Duysens and J. Amesz, Biochem. & Biophys. Acta, 24, 19 (1957). (13) L. N. M. Duysens a7d G. Sweep, Biochem. & Biophys. Acta, 25, 13 (1957). (14) J. M. Olsen, in “The Photochemical Apparatus, Its Structure and Function.” Brookkaoen Symo. Biol.. 11. 316 (1959). (15) E. Sawicki,’T.W. Stmely, J: D; Pfaff, and‘W. C.‘Elbert, Anal. Chem. Acta, 31, 359 (1!)64). (16) S. Udenfriend, “Fluorescence Assay in Biology and Medicine,” Academic Press, New York, 1962, p. 92. (17) B. L. Van Duuren, J . Phys. Chem., 68,2544(1964). (18) B. L. Van Duuren and C. E. Bardi, ANAL.CHEM.,35, 2198 (1963).
the necessary geometric arrangements, and a satisfactory theoretical background, methods that employ front face optics for quantitative analysis have not previously been developed, with the exception of a report (11) that the intracellular concentration of reduced pyridine nucleotide (NADH) in rat heart correlated with the intensity of NADH emission from the surface of the rat heart. This study covered a much narrower concentration range than those reported here. The methods reported here for three biologically important fluors have sensitivities and ranges comparable to or greater than existing methods. They are generally more rapid and simple than fluorescence methods employing right angle optics and eliminate the)edious extractions, purifications, and possible losses that may be involved in the preparation of optically clear solutions. The method is particularly well suited for analysis of fluorescent or potentially fluorescent molecules in preparations of biological origin.
RECEIVED for review January 3, 1967. Accepted April 17, 1967. Work supported by USPHS Grant No. Ca-08310.
Thin Layer Chromatography of Brominated Salicylanilides on Diethylaminaethyl Cellulose Norman E. Skelly AnaIytical Development Laboratory, The Dow Chemical Co., Midland, Mich.
48640
Kenneth A. Kamke Pitman-Moore Division, The Dow Chemical Co., Indianapolis, Ind. 46206
THEBROMINATED SALICYLANILIDES are becoming increasingly important because of their bacteriostatic properties. When incorporated into such v1:hicles as soaps and cleaners, growth of bacteria is inhibited on the surfaces to which they are applied. This activity is well documented in the literature (1-4). Also synergistic effects (5,6 ) have been observed when the brominated salicylanilides are used, in conjunction with other bacteriostats. Of the various brominated salicylanilides, 3,4’,5-tribromosalicylanilide is attributed to have the most activity. However, some activity is observed for 4’,5-dibromosalicylanilide and 2 ’,3,4’,5-tetrabroriio:;alicylanilide,while 3,5-dibromosalicylanilide is considered to be inactive. Two products are available under the registered trade names Tuasal 85 and Tuasal 100, (Dow Chemical Co., Midland, Mich.). The latter product is essentially pure 3,4’,5-tribromosalicylanilide, while the former is a mixture of approxi(1) Willard M. Bright, (to Unilever N.V.) Ger. Patent 1,126,058 (Cl. 23e), (Mar. 22, 1962); C.A., 57, 981 (1962). ~ Specialties, 39, (3,75 (1963). (2) Herbert C. Stecker, S O GChem.
(3) Unilever Ltd., Brit. Patcnt 965867 (Cl. A 61 l), (Aug. 6, 1964); C.A., 61, 12140 (1964). (4) R. C. S. Woodroffe, J. Hyg., 61, 283 (1963); C.A., 60, 7381 (1964). ( 5 ) Procter and Gamble Co , Belg. Patent 618650, (Sept. 28, 1962); C . A . 58, 5459 (1963). (6) Herbert H. Peller and William E. Jordan (to Procter and Gamble Co.) Ger. Patent 1,158,216 (Cl. A 51 1) (Nov. 28, 1963), C . A . , 60, 11859 (1964).
mately 15 % 3,5-dibromosalicylanilide and 85 % 3,4’,5-tribromosalicylanilide. Both products are prepared by the bromination of salicylanilide in a suitable solvent. Because impurities such as 4’,5-dibromosalicylanilide and 2‘,3,4’,5tetrabromosalicylanilide may be present, a method of analysis for this material was necessary. An ion exchangeultraviolet procedure (7) is available for determination of 4 ’,5-dibromosalicylanilide. However, resolution of the higher brominated salicylanilides would require a considerable volume of eluent and therefore an extended period of time. Because the brominated salicylanilides exhibit very intense fluorescence (sensitivity 0.1-0.2 pg), a thin layer chromatography (TLC) separation followed by examination under ultraviolet radiation would provide a very sensitive and fast method. Several TLC adsorption and partition methods on silica gel and alumina were tried without success. Because the brominated salicylanilides separate nicely by ion exchange, it was reasoned that a thin layer adsorbent having ion exchange properties might be a possible solution. This led to the use of diethylaminoethyl (DEAE) cellulose. EXPERIMENTAL
Apparatus and Reagents. Thin layer plates, 20- X 20-cm, were coated to a thickness of 375 microns using 300 G/DEAE cellulose (Mackerey, Nagel and Co., Duren, Germany, (7) N. E. Skelly, ANAL.CHEM., 35,1680 (1963). VOL. 39, NO. 8 , JULY 1967
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. Table I. R, Values for Brominated Salicylanilides on 375micron DEAE Cellulose R, value, acetic acid in methanol, 0 5 15 50 Salicylanilide 0.37 0.74 0.81 0.88 4 ’-Bromosalicylanilide 0.26 0.71 0.80 0.88 5-Bromosalicylanilide 0.07 0.70 0.76‘ 0.88 0.04 0.65 0.74 0.88 4’,5-Dibromosalicylanilide 3-Bromosalicylanilide 0.01 0.61 0.72 0.88 3,5-Dibromosalicylanilide 0.00 0.25 0.41 0.88 3,4’,5-Tribromosalicylanilide 0.00 0.17 0.31 0.83 2’,3,5-Tribromosalicylanilide 0.00 0.12 0.22 0.87 2’,3,4‘,5-Tetrabromosalicylanilide 0 .OO 0.07 0.13 0.79 0.00 0.06 0.11 0.59 Salicylic acid 5-Bromosalicylic acid 0.00 0.02 0.04 0.35 0.00 0.00 0.01 0.11 3,5-Dibromosalicylicacid
distributed by Brinkmann Instruments, Inc., Westbury, N. Y . )and a Desaga spreader. Ten grams of G/DEAE cellulose and 90 ml of water were homogenized in a 250-1111 beaker with a three-blade propeller attached to a mechanical stirrer at approximately 1700 rpm. The use of a Waring Blendor is not recommended because a large amount of air bubbles are incorporated into the slurry which leaves a large number of minute voids in the adsorbent surface upon drying. After air dryirig overnight, the plates were placed in an oven at 50’ C for 1 hour and finally stored in a desiccator until used. After thin layer separations, plates were examined in a Chromato-Vue (Ultra-Violet Products, Inc., San Gabriel, Calif.) ultraviolet box having both a long (360 mp) and short wavelength (253.7 mp) ultraviolet radiation sources. Standard brominated salicylanilide samples were prepared by condensing the proper bromosalicylic or salicylic acid with the correct bromoaniline or aniline compound to produce an unambiguous product. Melting point comparisons with literature values that were available showed satisfactory agreement. These were as follows: salicylanilide 135” C ; 4’bromosalicylanilide, 175-6.5 O C ; 5-bromosalicylanilide, 2401 ’ C; 4’,5-dibromosalicylanilide, 238.5 O C; 3-bromosalicylanilide, 149.5” C; 3,5-dibromosalicylanilide, 225 O C ; 3,4’,5tribromosalicylanilide, 227.5’ C; 2’,3,5-tribromosalicylanilide, 180-80.5 ’C ; 2’,3,4’,5-tetrabromosalicylanilide, 211-12’ C ; salicylic acid, 159’ C; 5-bromosalicylic acid, 169-70” C ; and 3,5-dibromosalicylic acid, 228-9 O C . Bromine content also gave a satisfactory comparison with calculated values. ACS grade methanol and C P grade acetic acid were used without further purification. Methanol-acetic acid mixtures were prepared on a vjv basis. An extremely high purity 3,4‘,5-tribromosalicylanilidewas prepared for use in the analytical standards. A sample containing 0.1 of the known impurities was recrystallized three times from 4 : l dioxane-water and dried at 100” C for 1 hour. Procedure. A sample of 3,4,’5-tribromosalicylanilide weighing 0.25 gram was placed in a 10-ml volumetric flask, dissolved with acetone, and diluted to volume. With the aid of a template and 10-p1 Hamilton syringe (No. 701), 4-pl aliquots were spotted at 1-cm intervals. The solution was added in increments so as to keep the spot size no larger than 3 mm in diameter. Standards containing 0.0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0% of the respective compounds, 4’,5 dibromo-, 3,5-dibromo- and 2’,3,4’,5-tetrabromosalicylanilide were added to the proper amount (250 mg or less) of purified 3,4’,5-tribromosalicylanilide in 10-ml volumetric flasks. These were diluted to volume with acetone and spotted among the samples. The plate was then placed in the developing tank with methanol/acetic acid (19 :I). Sep-
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ANALYTICAL CHEMISTRY
-
1.0.~
Solvent Front
0.8
0.6
t
m0.41
fi
3,5-Dibromosalicylanilide
0
21,3,4’,5-Tetrobramosalicylonilide
Origin
0.01
Figure 1. TLC of brominated salicylanilides on 375c( G/DEAE cellulose with 19:1 methanol-acetic acid aration was made by ascending development at ambient temperature. When the solvent front had traveled approximately 14 cm, the plate was removed from the tank and airdried. It was viewed under a long wavelength (360 mp) ultraviolet lamp. A visual comparison of the standards and samples was made and the concentrations of impurities were recorded. RESULTS AND DISCUSSION
R t values for the brominated salicylanilides and bromosalicylic acids are recorded in Table I. The compounds follow the same separation order on DEAE cellulose as on anion exchange resins (7); the weaker the acid, the larger the Rf value and the smaller the retention volume. Because of a secondary solvent front due to the acetic acid in the developing solvent, R, values for the lower brominated salicylanilides are always much less than unity. As noted in Table I the lower brominated salicylanilides have very similar R I values when acetic acid is present in the developing solvent. However, by first irrigating the plate with methanol, this group of compounds can readily be separated. By employing multiple development, further resolution is possible. In the analysis of a sample of unknown composition, a methanol development is recommended to ascertain the identity of the lower brominated salicylanilides that might be present. Because salicylic acid is a possible impurity in salicylanilide, the bromination products 5-bromosalicylic and 3,5-dibromosalicyclic acids are possible impurities in 3,4’,5-tribromosalicylanilide. With a 5 % acetic acid-methanol irrigation solvent, these impurities will remain at or near the origin. However, by using 50 acetic acid-methanol these acids can readily be resolved. Two-dimensional TLC was considered for this separation, but no advantage is gained by such a procedure. The detection method is nondestructive and does not change the components in any way; successive development with different methanol-acetic concentrations achieves the same results. It also allows the examination of 12 to 15 samples on one plate as compared with 1 by two-dimensional method. Initial experimentation in the analysis of 3,4’,5-tribromosalicylanilide presented the problem of localized tailing in the chromatogram. The difficulty was overcome by using a
z
template instead of a sharp pencil to mark the point of sample application. Apparently the sample became layered in the depression and failed to reach equilibrium conditions with the developing solvent. The chromatogram f’or the analysis of Tuasal bacteriostat is illustrated in Figure 1. Methanol/acetic acid (19:l) was the best solvent system for the determination of all three impurities using a singlc development. A smaller acetic acid concentration would fail to resolve 3,5-dibromosalicylanilide and 2 ’,3,4 ’,5-tribromosiilicylanilide, while a greater concentration of acetic acid would cause the 4’,5-dibromosalicylanilide to proceed into the secondary solvent front, reducing spot uniformity. Because 2 ’,3,5-tribronosalicylanilide has an R f value near that of 3,4’,5-tribrornci- and 2’,3,4’,5-tetrabromosalicylanilide, it would not be detected using the 19:l methanol/acetic acid solvent system. However, by multiple development with this solvent system or a moderately higher one, resolution of this impurity can be realized. A comparison of thc TLC method with that of the ion exchange procedure for the determination of 4’,5-dibromosalicylanilide is given in Table 11. The ion exchange method by its very nature has Iexcellent accuracy and precision and would be considered the standard method in this comparison. Generally the thin layer results agree to within =kO.lxabsolute of the amount present in the 0.1 to 0.5 range.
x
Table 11. Comparison of 4’,5-Dibromosalicylanilide Determination in 3,4’,5-Tribromosalicylanilide Ion exchangeultraviolet method ( 7 )
