Ion Exchange Separation of Brominated Salicylanilides and Hydroxybenzoic Acids NORMAN E. SKELLY and WARREN 8. CRUMMETT Special Services laboratory, The Dow Chemical Co., Midland, Mich.
b The application of nonaqueous ion exchange chromatography to hydroxybenzene derivatives has been extended to cover two new groups of compounds. Mixtures of brominated salicylanilides or of benzoic acid and hydroxybenzoic acid isomers are quantitatively adsorbed on Dowex 2-X8, ion exchange resin, acetate form. Gradient elution with acetic acidmethanol mixtures selectively desorbs the compounds. Concentrations are determined by ultraviolet spectrophotometry. Salicylanilide, 4‘-bromosalicylanilide, 5-bromosalicylanilide, and 4’,5-dibromosalicylanilide are separated by this method. The 3.5dibromo- and 3,4‘,5-tribrornosalicylanilide are not resolved. Benzoic acid and the three isomers of hydroxybenzoic acid are also separated by the above procedure.
I
years, brominated salicylanilides have become increasingly important because of their germicidal activity. When added to soaps, they reduce greatly the growth and metabolism of skin flora which give rise to the so-called body odor. Brominated salicylanilides are usually prepared by the bromination of salicylanilide. The choice of conditions depends on the desired product. In most instances, the product is a mixture and marketed as such. It therefore was necessary to develop methods of analysis for such mixtures. Infrared measurements are generally satisfactory for the determination of the major components in brominated salicylanilide mixtures. However, this technique lacks the necessary sensitivity for the determination of minor components. It is in this area that the ion exchange method has been found extremely useful. Sonaqueous ion exchange chromatography has found increasing use in rerent years (9,Ib, 18). l‘hus compounds having even very slight mater solubility may be earily separated. The use of continuous gradient elution ( I , 2 ) is often helpful in the separation of compounds that are not resolvcd by stepwise elution. Techniques for the separation of chlorophenols ( 1 4 ) linve been extended to the brominated sdiN RECENT
‘1680
ANALYTICAL CHEMISTRY
cylanilides and hydroxybenzoic acids. Chromatograms were monitored with an ultraviolet scanner having a 290-nip filter. The separation of the ortho, meta, and para isomers of hydroxybenzoic acid has been of interest for many years. Kumerous papers (3, 4, 6, 7, 11) have been published on this separation by paper chromatography. Liquid partition chromatography (6, 10) with silicic acid as the adsorbent has also been used. The 0- and m-hydroxybenzoic acids may be determined by their fluorescence spectra (16). The proposed ion exchange method separates benzoic acid and the three isomers of hydroxybenzoic acid. EXPERIMENTAL
Apparatus and Reagents. The gradient elution equipment, resin preparation, ultraviolet monitoring apparatus, and techniques have been dcscribed (14). I< R 0 h l I S.4T E D s A L I C Y L A NI L I D E S. These \\ ere prepared by condensing aniline or p-bromoaniline with the proper hromosalicylic acid so as t o give an unambiguous product. Melting points were comparable with those reported (8). A 13- X 400-rnin. chromatographic tube was used for some of the separations. 3,4 ’,5-Tribromosalicylaiiilide. For mixtures containing predominantly 3,4’,5-tribromosalicylanilide, dissolve a 0.1-gram sample by warming in 50 ml. of methanol. Adsorb the mixture on a 13- x 150-mm. bed of Dowex 2-X8, ion exchange resin, acetate form, 200 to 400 mesh. Connect the column to the gradient elution equipment and proceed with a 0.2Y0 acetic acid-methanol gradient elution. After salicylanilide arid 4’-bromosalicylanilide are removed as shown in Figure 1, continue the gradient elution with 2% acetic acidmethanol solution. Do not change the solution in the mixing flask for this second elution. When the 5bromosalicylanilide and the 4’,5-dibromosalicylanilide are completely eluted, terminate the gradient elution. Attach a reservoir to the column and remove the 3,j-dibromo- and/or 3,4’.5-tribromosalicylanilide by elution with 300 ml. of glacial acetic acid. Determine concentrations of the components in the rluates 1,s u1tr:iviol~t spcctrophotomctry.
4’,5-Dibromosalicylanilide. When 4’,5-dibroniosalicylanilide is the major component, adsorb a 10-nig. sample on a 13- X 330-mm. resin bed. The minor components will be 5-bromosalicylanilide and 3,4’,5-tribromosalicylanilide with possible traces of salicylanilide and 3,5-dibromosalicylanilide. Proceed with a 4oJ, gradient elution. Salicylanilide will emerge between 170 and 260 ml. of eluate; 5-bromosalicylanilide, between 500 and 750 ml.; and 4’,5 dibromosalicylanilide, between 750 and 1250 ml. Remove the remaining components as described above. HYDROXYBENZOIC ACIDS. Adsorb a 0.05- to 0.1-gram sample of m-hydroxybenzoic acid on a 13- X 330-mm. bed of the ion exchange resin from a small amount of methanol. Proceed with a gradient elution of 15% acetic acidmethanol solution. After the benzoic, p-hydroxybenzoic, and m-hydroxybenzoic acids are removed as shown in Figure 2, terminate the gradient elution. Remove any salicylic acid remaining on the resin by elution with glacial acetic acid. The salicylic acid will emerge between 100 and 300 nil. of the glacial acetic acid eluate. DISCUSSION
The brominated salicylanilides are separated on the basis of their acidity. The weakest acid is eluted from the ion exchange column first. Acidity of the brominated salicylanilides depends on the number and position of the substituted bromine atoms. Substitution on the salicylo ring contributes much more to the ionization of the hydrogen ion of the hydroxyl group than substitution on the prime ring. Therefore, although 4’,5dibromosalicylanilide has two bromine atoms as compared with one for 5-bromosalicylanilide, the dibrominated compound is only slightly more acidic than its monobromo counterpart. Analogously, 4’-bromosalicylanilide is slightly more acidic than salicylanilide and 3,4’,5tribromosalicylanilide than 3,5-dibromosalicylanilide. However, salicylanilide, 5-bromosalicylanilide, and 3,5dibromosalicylanilide show a considerable difference in acidity as a group, as do the structures 4’-bromosalicylanilide, 4‘,5-dibromosalicylanilide, and 3,4’,5-tribromosalicylanilide. In the separation of any unknown mixture, the ion exchange separation
--,~,--
80 Mg r n - H Y D R O X Y D E N Z O I C A C I D
GRADIENT ELUTION 13r153 m m DOWEX 2 A C E T A T E FORM ~
7 -1 1- -___ ~0 2 % Acetic A c i d - 4
SALICYLANILIDE
13 I 3 3 0 nim L O W E X 2 A C E T A T E FORM 15% A C E T I C A C I D - M E T H A N O L G R A D I E N T E L U T I O N
.1 ~__[ -
2 % Acetic Acid -I
1
SALICYLANILIDE
V O L U M E OF E L U A T E IN L I T E R S
-
IO
gradient elution
should be run, using conditions that give the best possible resolution. This will include the use O F the continuous gradient elution procedure. However, once the composition of the mixture has been ascertained, the elution procedure can be modified to fit a certain sample composition as a control analysis. Salicylanilide and b-bromosalicylanilide can be separated from 3,5-dibromosalicylanilide and/or 3,4‘,5-tribromosalicylanilide by stepwise elution with k e d concentrations of acetic acid in methanol. After adsorption of the mixture on a 13- x 150-mm. bed of the resin, the column is eluted with 100 ml. of O.3y0 acetic acid-methanol Eolution. This is followed by 100 ml of 3% acetic acid solution to remove salicylanilide and 200 ml. of 20% solution to remove 5-bromosalicylanilide. Highly brominated salicylanilides are removed with 300 ml. of glacial metic acid. 4‘Bromosalicylanilide and 4’,5-dibromosalicylanilide may be separated by the same procedure by substituting 0.5 and 5% acetic acid f;olutions for the 0.3 and 3y0 solutions, respectively. The salicylanilidA’-br omosalicylanilide and 5-bromosalicylanili de-4’,5-dibromosalicylanilide pairs, however, must be separated by the continuous gradient elution technique. The adsorption and desorption of the brominated salicylrtnilides are quantitative, as illustrated in Table I. Mixtures 1 and 2 were separated by continuous gradient elution, whereas mixtures 3, 4, and 5 were separated by stepwise elution. If there is an appreciable concentration of 5-bromosalicylrmilide in samples that are predominantly 4’,5-dibromosalicylanilide, the sample size should be reduced to prevent overlap of the chromatograms. However, some overlap can be tolerated, because the ultra-
violet sensitivities ( A per mg.) of the two compounds are almost identical. The ultraviolet spectra for the brominated salicylanilides are characterized by two absorption maxima (Table 11). The pairs of compounds having identical absorption maxima above 300 mp can be easily confused even after separation into pure fractions. Besides their position of elution from the column, a further identification method consists of determining the
Table 1.
Mixture 1
2 3 4
5
ratio of the absorbances of the two maxima. As shown in Figure 2, the isomers of hydroxybenzoic acid can be separated from each other and benzoic acid. Salicylic acid, although not shown on the chromatogram, is removed with glacial acetic acid after removal of the other acids. With the hydroxyl group in the ortho position, salicylic acid is a considerably stronger acid than its isomers and benzoic acid. The meta and para isomers are similar in their acidity, as shown in Table 111. Thus
Analysis of Known Mixtures of Brominated Salicylanilides Saiicyl4’,53,s3,4’,5-
anilide, 4‘-Bromo-, 5-Bromo-, Dibromo-, Dibromo-, Tribromo-, mg. mg. mg. mg. mg. mg.
Added Found
1.02 1.02
1.02 0.92
0.95 0.92
0.99
Added Found
1.02 0.98
1.02 0.91
0.95 0.91
0.99 0.87
1.00
Added Found
2.03 2.00
1.98 2.02
Added Found
2.03 2.05
2.49 2.62
Added Found
Table II.
1.96 1.91
1.90 1.99
2.04 2.06
96.9 96.1
Ultraviolet Absorption Data of Brominated Sa licylanilide in 5% Acetic Acid-Methanol Solution at 25’ C.
Compound Salicylanilide 4’-Bromosalicylanilide 5-Bromosalicylanilide 4’,5-Dibromosalicylanilide
Absorption max., mfi
303 302 316
Molar Absorption Molar absorpt,ivity max., mp absorptivity 8,380 268 11,100 11,500 274 15,700 7,200 272 10,000
3,5-l)ibromosalicylanilide
316 328
9,030 8,080
276 277
14,000 8,830
3,4’,5-Tritromosalicylanilide
328
9,350
2 80
12,200
VOL. 35, NO. 1 1 , OCTOBER 1963
0
1681
Table 111. pK. for Benzoic and Hydroxybenzoic Acids
Acid Benzoic p-Hydroxybenzoic m-Hydroxybenzoic o-Hydroxybenzoic
PK 4.20 4.48 4.08 2.97
the ion exchange column before either of these compounds. Other factors such as molecular configuration, adsorption, solubility, etc., besides p H probably have some effect on the separation. Other acids having pK, values greater than 3 should be separated by this technique. ACKNOWLEDGMENT
their separation is much more difficult. The separation of 0.8 mg. of p-hydroxybenzoic acid from 78.4 mg. m-hydroxybenzoic acid is a severe test for this separation. If more nearly equal quantities were ion exchanged, the separation would be much greater. Although benzoic acid has a pK, value between that of p-hydroxybenzoic and m-hydroxybenzoic, it emerges from
The aiithors thank T. E. Majewski arid G. C. AIattson for preparation of the lmminnteci snlicvlanilides.
( 3 ) Cats, H., Onrust, H., Chem. W e e k blad 54,456 (1958). (4) Cooke, A. C., New Zealand J . S c i 1 , 412 (1958). ( 5 ) Halver, C. V. D., J . Assoc. OBc. Agr. Chemists 43,593 (1960). ( 6 ) Joux, J. L., Ann. Fals. Fraudes 50, 205 (1957). (7) Lederer, M., Australian J . Sci. 1 1 , 208 (1949).
(8) Lemaire, H., Schramm, C. H., Cahn, A., J . Pharm. Sci. 50,831 (1961). ( 9 ) Logie, D., Analyst 82,563 (1957). (10) Marvel, C. S., Rands, R. D., Jr., J . Am. Chem. SOC.72,2642 (1950). (11) Mitchell, L. C., J . Assoc. O j i c . A g r . Chernzsts 40,592 (1957). (12) Plapp, E. W.,Casida, J. E 4 ~ ~ 1 , . CHEM30,1622 (1958). ( 1 3 ) Shelley, R. S . , Uniberger, C. J., ~
LITERATURE CITED (
S., Williams, R. J. R., Tiselius, A., Acta Chem. Scand. 6 ,
1) Alm, R.
826 (1952). (2) Busch, H., Hurlbert, R. B., Potter, V. R., J . Biol. Chem. 196,717 (1952).
Zbid., 31,593 (1959). (14) Skelly, N. E.,Ibzd., 33, 271.(1961). (15) Thommes, G. A., Lernmger, E., Ibid., 30,1361 (1958).
RECEIVED for review March 18, 1963. hccepted July 10, 1963.
Quantitative Analysis of Aspirin, Phenacetin, and Caffeine Mixtures by Nuclear Magnetic Resonance Spectrometry DONALD P. HOLLIS Spectroscopy Applicationslaboratories, Instrument Division, Varian Associates, 6 1 7 Hansen Way, Palo Alto, Calif.
b A procedure is described utilizing NMR spectrometry in the rapid quantitative analysis of mixtures of aspirin, phenacetin, and caffeine. Several known mixtures have been analyzed as well as some commercial preparations. The average deviations from the correct results were: aspirin, 1.1 %; phenacetin, 2.2%; and caffeine, 3.2%. The time required for the analysis is about 20 minutes.
C
has been dpvoted to the problem of developing suitable methods for the routine quantitative analysis of commercial analgesic preparations containing aspirin, phenacetin, and caffeine (APC). Procedures have been published utilizing separation by extraction, partition chromatography, and ultraviolet, visible, and infrared spectrophotometry. Conners cites a number of references on this subject (1). The method of the National Formulary (2) is of the extraction type but is not suitable for routine quality control work because of the time required to complete the analysis. Of the other procedures the infrared method of Parke et al. (3) seems to be most advantageous in terms of accuracy and speed. ONSIDERABLE EFFORT
1682
ANALYTICAL CHEMISTRY
Nuclear magnetic resonance spectrometry (NMR) can also be used very conveniently to analyze APC mixtures. The speed and accuracy of the S M R method to be described is about the same as that of other spectrometric methods, but it has the advantages of being more direct in the sense that a separate analytical peak of known origin is present for each component and that no calibration curves are required since the absorptivities for the protons giving rise t o the various analytical peaks are constant and unaffected by solvent or solute interactions. I n common with other spectrometric methods, no separation of the components is required for NMR analysis. The basic principle which permits the use of magnetic resonance absorption as a quantitative measure of a particular substance is that the signal strength is proportional to the number of magnetic nuclei. The dependence of the signal intensity on the relaxation times TI and Tz,the intensity of the driving radio frequency field, and the field sweep rate have been discussed in the literature (6, 6). I t should be remembered that the signal strength is also inversely proportional to the absolute temperature. In the present work the fact that the instrumental conditions chosen for the analyses are such that the
integrated intensities of the peaks employed are proportional to the number of protons present was demonstrated by the accuracy with which the composition of known standard mixtures could be determined. EXPERIMENTAL
Apparatus. Spectra were obtained a t 60 mc. per second using the Varian A-60 analytical KMR spectrometer, and in one case a t 100 mc. per second using the T’arinn HR-100 NMR spectrometer. Procedure. Weigh accurately into an A-60 NMR sample tube about 60 mg. of the carefully powdered sample. Using a micropipet, introduce exactly 0.500 ml. of CDClJ into the tube. It is important to avoid exceeding the solubility of aspirin which is least soluble of the three components. Cap the sample tube and shake, and gently warm the sample to effect complete solution of the APC. Binder materials such as starch and lactose will not dissolve but they do not interfere with the analysis and can be left in the sample tube. The sample is placed in the -4-60 spectrometer and the YAIR spectrum is obtained, the instrument gain, RF field, and sweep rate being adjusted to give a convenient integral presentation. The integral of each peak of interest should be run several times and the average
