High-Performance Liquid ChromatographicSeparation 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 conventional 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 suitability of I-IPLC for detection of trace amounts of optical
impurities in the case of r
[7]. Resolution 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) demonstrate 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-Camphorsulfonamides 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 m m 3 .

Results
Data for amide separations are reported in Table I. The
degree of separation is reported in each case as a (uncorrected). 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

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

Amine

I
5
MINUTES

o'

10

Fig. 1
9 HPLCseparation of (+-)-m-methoxy-~-methylbenzylamineas

the (+)-lO-camphorsulfonamidediastereomers; solvent system

(b).

a-methylbenzyl
p-methoxy-a-methylbenzyl
m-methoxy-c~-methylbenzyl
o-methoxy-c~-methylbenzyl
a-methylphenethyl
o-methyl-~-methylphenethyl
p-methoxy-~-methylphenethyl
p-chloro-c~-methylphenethyl
3,4-methylenedioxy-~-methylphenethyl
~ethylphenethyl
1-methyl-3-phenylpropyl
1-methyl-2-phenoxyethyl

(a)
1.60
1.54
1.00
1.31
1.24
1.19
1.22
1.25
1.24
1.12
1.13

(b)
1.56
1.56
1.71
1.28
1.04
1.00
1.00
1.08

1.07
1.00
1.08
No separations

(c)
1.59
1.00
1.73
1.13
1.10
1.05
1.03
1.15
1.12
1.00
1.05

(a) 8 % (9:1 CH2Cl2-isoptopanol)in isooctane at 2 cm3 min-1

(2000 psi)
(b) 5 % (9:1 CH:~Cl2-ethanol)in hexane at 1.5 cm3 min-1 (I100 psi)
(c) 15 % (9:1 butyl chloride-isopropanol) in hexane at 2 cm3 min-t
(1500 psi)

Discussion
Structures of the amines examined as (+)-lO-camphorsulfonomides 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-methylbenzylamine enantiomers have been observed by gas chromato-

ICH3

CH3

H
net~ylbenzylaratne

H
tx methvlphenelh~lamme

.iCH2CH3

H
a elhyIphenelhylamme

,c.~

~,.3
H
p methoxy c( methylbonzylamlne

CH3
H
o melhyI r rnethylphenethylarnme

.c.,
I melhu 3 phenylp~0pylamme

CH30--<~CH~--'Cm--N H2
/ H

OCH~
m melh~xy c( melhyIbenlylamm~

H
H
p melho~V a methylphenethyl3mlne t.melhyI 2 phenoxyelhylamlne
~
H 2 C--NH~
H
p chloto a rnethvlphenethylamme
CI-~C

0CFI~
0 melhoxvot n~elhylbenzylarmne

0~ . D - ~

CHe--'~IE--N
H
H3 H2

3,4 melhylenedloxya melhylphen~lhvtamme

GROUP A

GROUP B

GROUPC

Fig. 2

9 Structures of racemic amines separated as the (+)-10camphorsulfonamides

636

Chromatographia,
Vol. 9, No. 12, December 1976

graphy [8, 9, 11 ] and were attributed in part to the

close proximity of the aromatic ring to the hydrogenbonding amide group. While the mechanism of HPLC
separations such as those observed here is as yet unknown,
steric effects may well be contributing factors.
While there was essentially no variation in a in the three
solvent systems for the a-methylbenzylamine diastereomers,
separations of the methoxy positional isomers of o~methyl benzylamine showed a high dependence upon
the solvent system used. If the amide group is an important interaction site with the column, one might expect
good separations with the m- and p- methoxy derivatives,
while the o-methoxy would be poorer due to steric
hinderance. Only in the case of solvent system (b) was
this observed.
In the case of the amphetamines, the extra methylene
group between the chiral center and the ring seems to
yield poorer separations compared to a-methylbenzylamines. Addition of any of a variety of substituents of
the aromatic ring had only minor effects on a-values or
on retention times. Addition of another methylene
group to give 1-methyl-3-phenylpropylamine further
decreased a. Comparisons of separations for amphetamines (a-methylphenethylamines) to those for aethylphenethylamine show the former to be better
even though some steric bulk around the chiral center
was restored by the presence of an ethyl group rather
than a methyl. In the case of 1-methyl-2-phenoxyethylanaine no separations were obtained under any conditions.
This report describes the first extensive study of amine
enantiomer resolution by HPLC. It appears that HPLC
may be useful for both analytical and preparative
separations. Sample preparation time and analysis time
both were quite short for the amines examined here.
ShortCommunications

Acknowledgement

[4]

The author thanks Mr. Gerald M. Shkolnik of Varian Instrument

Division for technical assistance.

[51
[61

Literature
171
[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).

[8]
191
[10]
{ 111

D. Valentine, K. Chan, C. Scott, K. Johnson, K. Toth,

and G. Sancy, J. Org. Chem. 41, 62 (1976).
H. Furukawa, E. Sakakibara, A. Kamei, and K. Ito,
Chem. Pharm. Bull. 23, 1625 (1975).
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
G. Helmchen and Ir Strubert, Chromatographia 7, 713
(1974).
R.W. Sourer, J. Chromatogr. 108, 265 (1975).
R.W. Souter, J. Chromatogr. 114,307 (1975).
P.D. Bartlett and L.H. Knox, "Organic Syntheses,"
Vol. 45, John Wiley and Sons, Inc., 1965 p. 14
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 1 9 7 6

Short Communications

637