S. J. Romano Colgate-Palmolive Company Piscataway, New Jersey 08854
Separation of Bromoaniline Isomers by Cation-Exchange Chromatography
V e r y little has been reported in the liters, tnre concerning the separation of o-, m-, and p-bromoaniline. Woolfolk, et al.,' have partially separated these isomers by chromatographing the p-phenylazobenzenesulfonyl chloride derivatives on 2: 1 silicic acidcelite columns. Gillio-Tos, et. a].,% have quantitatively separated o-, m-, and p-bromoaniline by thin-layer chromatography using dibutyl ether-acetic acid-nhexane as developing solvent. The possibility exists for separating these compounds by ion-exchange chromatography on Sephadex C-25 CM resin. This work deals with separation of p- from o- and/or mbromoaniline and optimum conditions for fast resolution.
-M*CW COLUMN
---- M I C R O COLUMN
Experimental Reagents. Eastman White Label m- and c-hromoaniline were a-Bmmoaniline was Eastman
ased without further ourification.
the manufacturer's recommendlttions. Chromatography Columns. A Sephadex K15/30 column (30 cm X 1.5 cm i.d.) was used and is herein designated as the macrocolumn. A Chemtronix microbore column (50 cm X 2.75 mm i d . ) was fitted with s. SV-8031 sample injection valve and used as the microcolumnin this work (Chemtronix, Berkeley, Cdifornia). The column effluentswere monitored by a Beckman Model D. U. spectrophatometer modified with a Spectral Energy Recording Attachment connected to a Sargent Multi-Range recorder. The operating wavelength was 260 mp and a Beckman 10 mm How cell was used (0.3 ml volume). The flow rate was controlled by a Milton Roy Minipump (St. Petemburg, Florida). The macrocolumn was packed with Sephadex C-25 slurried in 1 N hydrochloric acid to a height of 21 cm and washed with several bed volumes of 1 N hydrochloric acid. Distribution ratios were found to be 3.5,3.6, and 2.6 for c-, m-, and p-bromoaniline, respectively. A mixture of 0.02 mmole each in c- and p-bromoaniline was chromatographed on this same column. Flow rates were kept constant at 0.53 cm/min. The results are shown on the figure. Amixtureof 0.0056 mmole each in o- and p-bromosniline was chromatographed on the microcolumn with 1 N hydrochloric acid as eluent and a How rate of 4.0 cm/min. This separation is also shown in the figure.
Discussion
Speed and resolution are important considerations in any chromatographic separation. The macrocolnmn gives incomplete resolution and the time for complete elution was about 90 min. However, the microcolnmn WOOLFOLK, E. O., REYNOLDS, W. E., MASON, J. L., J . Or#. C h m . , 24,1445-50 (1959). A., J . ChromaGILLIO-TOS. M.. PREYITERA, S. A,, VIMERCATI, tog., 13(2), 571-2 (1964). 1
478 / Journal of Chemiml Education
tMlN
Separation of a. m d p-bromoaniline with 1 N hydrochloric acid eluent. Broken line, 0.0056 mmole each a t Row rote of 4.0 cm/min on microcolumn. Solid line, 0.02 mmole each 01 flow rate of 0.53 cm/min on macmcolumn.
separates the compounds quantitatively in only 36 min. A complete separation could be obtained using a longer macrocolnmn but this would be done a t the expense of speed. Conditions which control the speed and resolution are column length, column diameter, flow rate, particle size, and sample size. I n analytical separations the best conditions are those of a long, thin column with fast flow, small particle packing, and a small sample. The table compares these parameters for this separation. It was anticipated that the high flow rate used in the microcolnmn would cause considerable tailing but this was not the case. In fact, the macrocolnmn was found to give more tailing. The chromatographic setup consisting of the injection system, microcolnmn and the detector lends itself to quantitative analysis of mixtures. The detector has been found to be stable for a t least 8 hr with little baseline drift. The injection system allows for repetitive sample injection without disturbing the flow. Comparison of Separation Parometers Column length Column diameter -Wlnw .... rnt,e .-.. Sample size Resin article size
Microcolumn
Macrocolumn
50 cm 2.75 mm 4.0 cmlmin 0.0056'mmole 40-120 p
21 cm 1 . 5 cm 0.53 cmfmin 0.020 mmole 40-120 p
