Resolution of (±)-β-methylphenylethylamine by a novel chiral stationary phase for Pirkle-type column chromatography

Article abstract of DOI:10.1002/chir.20736

In this study, a new Pirkle-type chiral column stationary phase for resolution of β-methylphenylethyl amine was described by using activated Sepharose 4B as a matrix, L-tyrosine as a spacer arm, and an aromatic amine derivative of L-glutamic acid as a ligand. The binding capacities of the stationary phase were determined at different pH values (pH = 6, 7, and 8) using buffer solutions as mobile phase, and enantiomeric excess (ee) was determined by HPLC equipped with chiral column. The ee was found to be 47%.

Full text of DOI:10.1002/chir.20736

CHIRALITY 22:252–257 (2010)
Resolution of (6)-b-Methylphenylethylamine by a Novel Chiral
Stationary Phase for Pirkle-Type Column Chromatography
1
HAYRULLAH YILMAZ,1 GIRAY TOPAL,1 RESIT CAKMAK, AND HALIL HOSGOREN2
*
1Department of Chemistry, Faculty of Education, Dicle University, Diyarbakir, Turkey
2Department of Chemistry, Faculty of Science and Arts, Dicle University, Diyarbakir, Turkey
ABSTRACT
In this study, a new Pirkle-type chiral column stationary phase for reso-
lution of b-methylphenylethyl amine was described by using activated Sepharose 4B as
a matrix, L-tyrosine as a spacer arm, and an aromatic amine derivative of L-glutamic acid
as a ligand. The binding capacities of the stationary phase were determined at different
pH values (pH 5 6, 7, and 8) using buffer solutions as mobile phase, and enantiomeric
excess (ee) was determined by HPLC equipped with chiral column. The ee was found
C
VV
to be 47%. Chirality 22:252–257, 2010.
2009 Wiley-Liss, Inc.
KEY WORDS: Sepharose 4B; resolution; chiral amines; HPLC; Pirkle-type column
chromatography
INTRODUCTION
It is a well-known fact that two important features of chi-
ral compounds make them distinctive from achiral com-
pounds: Firstly, their behavior toward plane-polarized light
and secondly, their interactions with other chiral com-
pounds. Chiral compounds are separated into enantiomers
through different chromatographic (HPLC, liquid chroma-
tography and Clay column chromatography) methods.1–3
In addition, the above-mentioned separation processes are
The separation of racemic mixtures of chiral compounds
also conducted through capillary electrophoresis method.4
If amines with high enantiomeric purity are of biological
importance, they can have wide medical applications. For
this reason, the preparation of chiral compounds and their
analysis is very important. High enantiomeric purity is
always demanded in environmental chemistry, pharmaceu-
tical industry, and clinical analyses. The main power
behind the design enantioselective reactions is the phar-
maceutical industry. Typically, only one enantiomer of a
chiral drug is active. Ibuprofen (sold under the names
Advil, Motrin, or Nuprin) which is anti-inflammatory is
such an example. Only the (S) isomer of ibuprofen is
active. The (R) isomer of ibuprofen does not show anti-
inflammatory effect.5
(S,S)-Ethambutol is one of the enantiomers of a chiral
tuberculostatic drug that especially shows effective ther-
apy, but (R,R)-ethambutol is toxic to humans. (S)-2-Amino-
butanol is the key intermediate in the manufacture of the
(S,S)-ethambutol.6
b-Methylphenylethylamine belongs to the group of
drugs related to amphetamines. It is known that it has
stimulant effects in man, as the b-methyl group slows me-
tabolism by monoamine oxidase enzymes. The structural
formula is given below:
is conducted by using different techniques at present. One
of these is chromatographic separations. The chiral sta-
tionary phases (CSPs) that are used for this process
include chiral ligand-exchange columns, chiral affinity col-
umns, helical polymer columns, cavity columns, and Pir-
kle-type columns.7 Polysaccharide derivatives, cyclodex-
trin derivatives, macrocyclic chiral compounds, ligand
exchange complex, crown ether, imprinted polymers, and
some molecular selectors such as Pirkle-type compounds
are used in the preparation of CSPs.8–12 The chiral selec-
tors used in CSPs include polysaccharide derivatives and
studied enantioseparation of binaphthyl compounds13 and
imidazolinone herbicides.14 Farkas et al.15 reported the
preparation and testing of a new pyridino-18-crown-6 ether
_
Contract grant sponsor: TUBITAK (The Scientific and Technological
Research Council of Turkey); Contract grant number: 106T724 (TBAG-
HD/224).
*Correspondence to: Giray Topal, Department of Chemistry, Faculty of
Education, Dicle University, Diyarbakır 21280, Turkey.
E-mail: gtopal@dicle.edu.tr
Received for publication 9 January 2009; Accepted 25 March 2009
DOI: 10.1002/chir.20736
Published online 3 June 2009 in Wiley InterScience
(www.interscience.wiley.com).
C
VV
2009 Wiley-Liss, Inc.

RESOLUTION OF ( )-b-METHYLPHENYLETHYLAMINE
253
based CSP and studied the enantioseparation of proto- stationary phase. Acetate and phosphate buffer solutions
nated primary arylalkylamines and aromatic amino acid were used as mobile phases at different pH values. In the
derivatives. Enantiomers of some amino acid ester deriva- determination of enantiomeric purity, perchloric acid was
tives have been separated on brush-type CSPs derived used as HPLC mobile phase.
from terpenoid compounds.16
Pirkle-type column chromatography is a technique
developed by the use of a series of different stationary
phases as a matrix formed by the amino acid derivatives
getting connected to the silica with covalent or ionic
attachment. Generally, N-(3,5-dinitrobenzoyl) phenylgli-
cyne is used as stationary phase. As these columns can be
obtained commercially, they can also be prepared for prep-
arative or analytical purposes. Mechanisms of enantio-
meric separation are explained with many combined fac-
tors such as interaction through charge transfer, hydrogen
bonding, dipole–dipole interactions, and steric effects.17,18
Pirkle et al. developed Pirkle-type CSPs prepared from
N-(3,5-dinitrobenzoyl) derivatives of (S)-leucine and (R)-
phenylglycine and used them to separate the enantiomers
of N-aryl-a-amino esters and 2-carboalkoxyindolines.19
Hyun et al.20 used Pirkle-type p-acidic CSP for the enantio-
separation of amide derivatives of naproxen. The enan-
tiomers of amino acid derivatives have been separated on
new Pirkle-type CSP with long alkyl chains.21 Hyun et al.22
developed a new Pirkle-type CSP derived from N-(3,5-dini-
trobenzoyl) leucine N-phenyl N-alkylamide and the resolu-
tion results were compared with those on various commer-
cial p-acidic CSPs and enantiomers of racemic 2-hydroxy-
carboxylic acids were resolved as their O-ethoxycarbonyl
p-basic anilide derivatives on a new CSP. In a study by
Kraml et al., a series of carbobenzyloxy (cbz) derivatives
of commercially available racemates was prepared and an-
alyzed by enantioselective chromatography using a variety
of mobile phases and polysaccharide and Pirkle-type
CSPs. The cbz-derivatized product consistently demon-
strated enhanced chiral resolution under HPLC and super-
critical fluid chromatography conditions.23
Apparatus
A 1.0 cm 3 20 cm (ID X L) Luer-lock, nonjacketed liquid
chromatography column (Sigma-Aldrich) was used in the
resolution of the chiral amine. Flow rate of eluates was
adjusted with Watson Marlow-323 peristaltic pump. Amine
concentrations in eluates were determined with a Perkin-
Elmer-k-35 UV-spectrophotometer. H and 13C NMR spec-
1
tra of synthesized compounds were recorded by Bruker AC
400 MHz NMR spectrometer. Infrared (IR) spectra were
recorded by Mattson 1000 FTIR-spectrophotometer. Enan-
tiomeric purity of samples was determined with Bio-Der
2100 HPLC dual-pump system and a Shimadzu-3001 UV de-
tector by using Crownpak CR-11 column (CR (1) 27014
chiral separation column and CR (1) 27011 guard column).
Synthesis of Ligand
N-p-nitrobenzoyl-L-glutamic acid (C12H12N2O7).
The L-amino acid was reacted with p-nitrobenzoylchlor-
ide and the product was reduced to the amine according
to standard methods.24,25
0.73 g (0.5 mmol) of L-glutamic acid was dissolved in
15 ml 1 N NaOH and 1.5 g sodium acetate was added.
Then, 0.93 g (0.5 mmol) powder p-nitrobenzoylchloride
was added into this solution. The purple solution was
stirred for 5 min and filtered. Then, 3 N HCl was added
dropwise until the filtrate was yellow. The solution contin-
ued to be acidified with dilute hydrochloric acid by con-
trolling pH. A white-yellow precipitate occurred. The mix-
ture was kept for 1.5 h at room temperature and filtered
several times in vacuum. The filtrate was kept for 15 min
and precipitate was formed. Thus, N-p-nitrobenzoyl-L-glu-
tamic acid was obtained. White product was filtered and
crystallized from ethanol. Yield: 0.94 g (64%), mp 1928C,
[a]3D0 5 215.78 (C 5 0.6, in ethanol).
We prepared a new CSP by using Sephorose 4B as a
matrix, L-tyrosine as a spacer arm, and the aromatic amino
derivative of L-glutamic acid as ligand. The spacer arm was
placed between the matrix and the ligand. We tried to sep-
arate b-methylphenylethyl amine into its enantiomers,
using buffer solutions at various pH values as mobile
phase from this column. Our aim in this study is to deter-
mine the separation capacity of the Pirkle-type column we
have prepared regardless of the characteristics of the
amine used.
1H NMR (CDCl3) d: 1.93–2.16 (m, 2H), 2.36–2.50 (m,
2H), 4.40–4.46 (m, 1H), 8.12 (d, 2H), 8.33(d, 2H), 9.02 (d,
1H), 12.35(b.s, 2H).
EXPERIMENTAL
Chemicals
13C NMR (CDCl3) d: 26.3, 30.9, 52.7, 124.0, 129.5, 140.0,
149.6, 165.5, 173.5, 174.2.
Sodium hydroxide, sodium acetate, L-glutamic acid, L-ty-
rosine, p-nitrobenzoyl chloride, hydrazine-hydrate solution,
Pd/C, b-methylphenylethylamine, activated Sepharose 4B,
perchloric acid, diethyl ether, and absolute ethanol were
purchased from Sigma and Merck (analytical grade,
Merck, Darmstadt, Germany) and Fluka (Switzerland). All
other reagents were analytical grade and used without fur-
ther purification. Activated Sepharose 4B was loaded as
N-p-aminobenzoyl-L-glutamic acid (C12H14N2O5).
Chirality DOI 10.1002/chir

254
YILMAZ ET AL.
Fig. 1. Preparation of chiral gel (Sepharose-4B-L-tyrosine-L-glutamic acid derivative).
Preparation of Chiral Column
The reaction was carried out in a 250 ml two-necked
round-bottom flask equipped with a reflux condenser and
a dropping funnel and placed in a magnetic stirrer. 0.8 g
N-p-nitrobenzoyl-L-glutamic acid was placed in the flask
and dissolved in 60 ml ethanol with stirring. 0.05 g Pd/C
was added to the stirred solution, and 10 ml hydrazine
hydrate solution was added dropwise to the mixture. The
reaction mixture was refluxed at 808C for 2.5 h. The hot
dark solution was filtered immediately and the ethanol
evaporated. Thus, reduced amine was obtained. Yield: 0.55
g (78%), [a]3D0 5 29.88 (C 5 0.8, in ethanol).
Affinity chromatography columns for enzyme purifica-
tion are prepared with Sepharose 4B matrix and different
enzymes specific ligands are used.26 By modifying this
method, we prepared a new stationary phase with N-p-ami-
nobenzoyl-L-glutamic acid as a ligand to interact with a chi-
ral amine and activated Sepharose 4B as a matrix.
Five grams of activated Sepharose 4B was suspended in
20 ml distilled water and washed on a Buchner funnel sev-
eral times with 200 ml distilled water to remove the impur-
ities and 100 ml 0.2 M NaHCO3 buffer (pH 10) at room
temperature, respectively. This mixture was placed into a
100 ml beaker and L-tyrosine solution (80 mg/20 ml dis-
tilled water) was added by stirring with a magnet for 2 h at
148C, and then left for 16 h without stirring at the same
1H NMR (CDCl3) d: 1.85 (d, 2H), 1.95–2.08 (m, 2H),
4.11 (m, 1H), 6.55 (d, 2H), 7.55 (d, 2H), 7.87 (d,2H). Car-
boxylic acid and amide protons were not observed.
13C NMR (CDCl3) d: 29.4, 34.3, 55.1, 113.2, 121.8, 128.9,
151.9, 166.2, 177.0, 179.2.
Chirality DOI 10.1002/chir

RESOLUTION OF ( )-b-METHYLPHENYLETHYLAMINE
255
(pH 6). The solution was packed into a column (1 cm 3
20 cm) and, after precipitation of the gel, equilibrated
with the same buffer using a peristaltic pump (flow rate:
30 ml/h). The reaction scheme is shown in Figure 1.
Resolution of b-Methylphenylethylamine
A solution of racemic b-methylphenylethylamine (100 ll
amine/5 ml 0.1 M phosphate buffer pH 6) was loaded onto
the column and eluted with phosphate buffer (pH 6) flow
rate 25 ml/h. Resolution studies were repeated with the
same buffer at pH 7 and 8. Elutes were collected in 3 ml
fractions, and absorbance values were determined at
256 nm by UV spectrophotometer. The amine was col-
lected in the 3rd, 4th, and 5th tubes for each buffer (pH 6,
7, and 8). The pH of the tubes was adjusted to approxi-
mately pH 14 with 1 N NaOH solution and extracted with
diethyl ether. The ether phase was evaporated and 2 ml
HClO4 (pH 1.5) was added to the residue in each tube.
This study was repeated for 50 and 25 ll racemic b-
methylphenylethylamine at the same conditions. The
amine was collected in the 7th, 8th, and 9th tubes with pH
6 buffer.
Fig. 2. Resolution of racemic 100 ll b-methylphenylethylamine mix-
ture at different pHs by the Sepharose 4B chiral Pirkle-type column.
temperature. It was then washed again on a Buchner fun-
nel with 100 ml 0.2 M NaHCO3 buffer (pH 8.8). Thus, L-ty-
rosine was coupled to activated Sepharose 4B.
Fifty milligrams of N-p-aminobenzoyl-L-glutamic acid
was dissolved in 10 ml 1 N HCl. Sodium nitrite solution
(100 mg NaNO2/5 ml distilled water) was added to this so-
lution slowly with stirring at 08C. The diazotized N-p-ami-
nobenzoyl-L-glutamic acid was poured into 40 ml of the
Sepharose-4B-L-tyrosine suspension. The pH was adjusted
to 9.5 with 1 M NaOH and the mixture stirred for 3 h at
room temperature. The yellow suspension was washed
with 500 ml distilled water and 200 ml phosphate buffer
Determination of Enantiomeric Purity by HPLC
Enantiomeric purity of eluates (Preparation of Chiral
Column section) was measured by chiral HPLC with a UV
detector. The amines were diluted with distilled water
(mobile phase: HPLC grade HClO4 pH 1.5), flow rate:
1.0 ml/min, column pressure: 100 bar, wavelength: 200 nm).
Fig. 3. The chromatogram of 100 ll racemic mixture of b-methylphe-
nylethylamine on CHIRALPAK1 CR-11 with 0.03 N HClO4 for 5th fraction
in pH 6 buffer (Flow rate: 1.0 ml/min, temperature: 258C, detection: UV
200 nm).
Fig. 4. The chromatogram of 50 ll racemic mixture of b-methylpheny-
lethylamine on CHIRALPAK1 CR-11 with 0.03 N HClO4 for 8th fraction in
pH 6 buffer (Flow rate: 1.0 ml/min, temperature: 258C, detection: UV
200 nm).
Chirality DOI 10.1002/chir

256
YILMAZ ET AL.
The ee values were 34.5, 47.1, and 24.2% for 100, 50, and
25 ll racemic mixtures, respectively. Enantioselectivity (a)
for sample of 50 ll was 1.30, which is smaller than others
under the same conditions. The chromatographic results
obtained with various initial amounts of racemic b-methyl-
phenylethylamine have been shown in Figures 3–5.
After each separation study conducted with different pH
values and different amine amounts, the column was
regenerated by washing with 1 N 100 ml HCl. Carboxyl
groups of L-glutamic acid, bound to the L-tyrosine at sta-
tionary phase, were turned into nonionic form. Then, it
was equilibrated with 100 ml 0.1 M phosphate buffer.
Thus, the column became ready for use.
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Chirality DOI 10.1002/chir