High-Performance Liquid Chromatographic Separation of Enantiomeric Amines

R.W. Sourer

The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46206, U.S.A.

Summary

Separations of twelve different racemic amines were studied by high-performance liquid chromatography (HPL C) in several column-solvent combinations. Relative retentions, which show some dependence upon the substitution at the asymmetric centers, are reported for the amines which were examined as the (+)- 10-camphorsulfonamides.

The separation of optical isomers by gas chromatography (GC) has been the subject of extensive recent research. Racemates of a variety of classes of compounds can now be resolved on chiral stationary phases or can be converted to a mixture of diastereomers for separation on conven- tional phases. Direct resolution techniques were recently reviewed by Lochmiiller and Souter [ 1 ] while diastere. omer separation methods have been reviewed by Gil-Av and Nurok [2].

Very little work has appeared using HPLC as a tool for analytical or preparative enantiomer resolution. Koreeda, Weiss and Nakanishi [3] used HPLC for preparative separation of some cis-diol enantiomers in an effort to establish the absolute configuration of natural (+)-abscisic acid. The ratios of enantiomers of citronellic and related acids were determined by Valentine et al. by HPLC as well as by NMR [4]. Furukawa et al. showed that several amino acid enantiomers were separable as diastereomers [5] after introduction of a p-nitro-benzyl moiety as a chromophor for detection. The resolution of diastereomeric peptides has also been examined [6]. Finally, Helmchen and Strubert demonstrated the suit- ability of I-IPLC for detection of trace amounts of optical impurities in the case of r [7]. Resol- ution by GC of the enantiomers of amphetamine and some related amines was recently reported by Souter [8, 9].

The present HPLC work was undertaken to (a) demon- strate the applicability of HPLC in optical isomer separation work, (b) demonstrate that relatively short sample preparation time is necessary because one need not introduce any special groups to improve volatility and (c) determine whether improved separations are possible

(as opposed to gas chromatography) based on some observations for certain peptides [6]. In addition, structural effects on resolution are of interest. This work examines the HPLC behavior of twelve different amines as the (+)-10-camphorsulfonamide diastereomers.

Sample Preparation

All amines (except for 1-methyl-2-phenoxyethylamine, which was purchased from Aldrich Chemical Co., Milwaukee, Wisconsin) were obtained in-house and were used without further purification. Some amines were obtained as salts which were treated with excess sodium hydroxide, extracted with ethyl ether, and dried over anhydrous sodium sulfate. No evidence of decomposition was noted for any of the samples, even after 2 weeks storage at room temperature. The (+)-10-camphorsulfonyl chloride was prepared on a 0.06 mole scale using the procedure of Bartlett and Knox [10]. The crude product demonstrated the correct NMR spectrum and was dried in vacuo over P2 os until use. (+)-10-Camphorsulfona- mides of the amines were prepared by the procedure of Furukawa [5] using 0.001 moles of amine and 0.001 moles of crude (+)-10-camphorsulfonyl chloride in diethyl ether (10 cm 3). The aqueous-ether mixtures were stirred vigorously at room temperature for one hour, were then acidified with 1 mole dm -a HCI, and were finally extracted with ether and dried over anhydrous sodium sulfate. The crude camphorsulfonamides were used "as is" after evaporation under dry nitrogen to volumes of 5 cm 3 .

Analytical Procedure

A Varian 8520 liquid chromatograph with a Variscan variable wavelength detector operating at 254 nm was used for all measurements. The column was a Varian Micropak-NH2-10/~ (25 cm X 0.2 cm id). Solvent systems, which are described with the data, were prepared from distilled-inglass or spectroscopically pure solvents. The flow rates and approximate operating pressures are described with the experimental results. Tile size of injections was 5 mm 3 .

Results

Data for amide separations are reported in Table I. The degree of separation is reported in each case as a (un- corrected). For the ~t-methylbenzylamine, a sample enriched in one isomer was used for all experiments to facilitate peak identification. All separations were achieved in 10 minutes or less. A typical chromatogram is shown in Fig. 1.

Chromatographia, Vol. 9, No. 12, December 1976 Short Communications 635

o'

Fig. 1

I 5

M INUTES

I 10

9 HPLC separation of (+-)-m-methoxy-~-methylbenzylamine as the (+)-lO-camphorsulfonamide diastereomers; solvent system (b).

Table I. Calculated a-values for amide separations on Micropak-NH2-10//

Amine 1 (a) I (b) t (c)

a-methylbenzyl 1.60 1.56 1.59 p-met hoxy-a-methylbenzyl 1.54 1.56 1.00 m-met hoxy-c~-methylbenzyl 1.00 1.71 1.73 o-met hoxy-c~-methylbenzyl 1.31 1.28 1.13 a-methylphenethyl 1.24 1.04 1.10 o-methyl-~-methylphenethyl 1.19 1.00 1.05 p-met hoxy-~-methylphenethyl 1.22 1.00 1.03 p-chloro-c~-methylphenethyl 1.25 1.08 1.15 3,4-methylenedioxy-~-methyl- phenethyl 1.24 1.07 1.12 ~ethylphenethyl 1.12 1.00 1.00 1-methyl-3-phenylpropyl 1.13 1.08 1.05 1-methyl-2-phenoxyethyl No separations

(a) 8 % (9:1 CH2Cl2-isoptopanol) in isooctane at 2 cm 3 min -1 (2000 psi)

(b) 5 % (9:1 CH:~Cl2-ethanol) in hexane at 1.5 cm 3 min -1 (I100 psi) (c) 15 % (9:1 butyl chloride-isopropanol) in hexane at 2 cm 3 min -t

(1500 psi)

Discussion

Structures of the amines examined as (+)-lO-camphor- sulfonomides by HPLC are shown in Fig. 2. Group A includes the ~-methylbenzylamines, Group B the amphetamines, and Group C some substituted related anaines.

Three different solvent systems were evaluated with the Micropak-NH2 column, and in all cases the largest a values were obtained with the a-methylbenzylamine compounds. Excellent separations of a-methylbenzyla- mine enantiomers have been observed by gas chromato-

ICH3 CH3 ~ .i CH2CH3

H H H net~ylbenzy laratne tx methvlphenelh~lamme a elhyIphenelhylamme

~,.3 ,c.~ .c.,

H CH 3 H I melhu 3 phenylp~0pylamme p methoxy c( methylbonzylamlne o melhyI r rnethylphenethylarnme

CH30--

Acknowledgement

The author thanks Mr. Gerald M. Shkolnik of Varian Instrument Division for technical assistance.

Literature

[11 C.H. LochmiillerandR. W. Souter, J. Chromatog. 113, 283 (1975).

[21 E. GiI.Av and D. Nurog in "Advances in Chromatography", vol. 10. J. Giddings and R. Keller, eds. New York: Marcel Dekker, Inc. 1974, pp 99-172

131 M. Moreeda, G. Weiss and K. Nakaniski, J. Am. Chem. Soc. 95,239 (1973).

[4] D. Valentine, K. Chan, C. Scott, K. Johnson, K. Toth, and G. Sancy, J. Org. Chem. 41, 62 (1976).

[51 H. Furukawa, E. Sakakibara, A. Kamei, and K. Ito, Chem. Pharm. Bull. 23, 1625 (1975).

[61 C.M. Deben and H. Joshua, "Chemistry and Biology of Peptides, Proceedings of the 3rd American Peptide Symposium", J. Maienhofer, Ed., Ann Arbor Science Publishers, 1972

171 G. Helmchen and Ir Strubert, Chromatographia 7, 713 (1974).

[8] R.W. Sourer, J. Chromatogr. 108, 265 (1975).

191 R.W. Souter, J. Chromatogr. 114,307 (1975). [10] P.D. Bartlett and L.H. Knox, "Organic Syntheses,"

Vol. 45, John Wiley and Sons, Inc., 1965 p. 14 { 111 C.H. Lochmi21ler and R. W. Souter, J. Chromatogr. 88,

41 (1974).

Received: April 22, 1976 Accepted: July 27, 1976

Chromatographia, Vol. 9, No. 12, December 1976 Short Communications 637